AN ABSTRACT OF THE
DISSERTATION OF
Ethel Owusuwaa Owusu for the degree of Doctor of Philosophy in Biochemistry and
Biophysics presented on June 28, 2004.
Title: Characterization of the Zea
mays ssp. mays
Abstract aPProveRedacted
TOUSLED-Like Kinases
for privacy
L (
-
carøiI Rivin
This dissertation describes the cloning and characterization of the TOUSLED-
like kinases genes of maize (ZmTLKs). The TOUSLED-like kinases (TLKs) are a
conserved family of nuclear Ser/Thr kinases in higher eukaryotes. The maize genome
has three TOUSLED-like kinase genes (ZmTLK1, ZmTLK2, and ZmTLK3). Based
upon sequence similarity, the ZmTLKs are divided into two classes, the ZmTLK1 and
the ZmTLK2/3 class. The origins of these genes can be inferred from their map
positions and relationships with TLKs in other Zea species. The ZmTLK1 and
ZmTLK2 genes occupy syntenous positions on chromosome arms 1L and 5S in the
maize genome. There are two equivalent classes of TLK genes in other Zea species,
altogether indicating that the two ZmTLK classes are orthologous genes from the
precursor species of maize, an ancient allotetraploid.
Gene expression studies of ZmTLKs show that there is a higher level of
expression in tissues undergoing DNA synthesis. This is consistent with studies of
TLKs in animal systems that show involvement in chromatin assembly/remodeling
activities during DNA replication and repair, as well as in transcription. The highest
level of gene expression for the ZmTLK2/3 class was observed during development of
the endosperm, in a period of massive nuclear endoreduplication. ZmTLK1 is not
upregulated in endoreduplicating endosperm, suggesting functional divergence
between the two classes of ZmTLK genes.
The function of the ZmTLKs was examined by testing whether maize TLK
genes could complement the tousled mutant of Arabidopsis. In Arabidopsis thaliana,
recessive mutations in the single copy TOUSLED (TSL) gene cause moderate
vegetative and severe floral defects, suggesting that TLKs may play a role in gene
expression modulation through chromatin remodeling. The ZmTLK proteins are 84%
identical to TSL in the catalytic region and 45 - 49% at the N-terminal regulatory
domain. However, structural features of the N-terminal region domains of the
ZmTLKs are similar to that of TSL. Arabidopsis tsl-1 mutant plants were transformed
with ZmTLK2, under the control of the CaMV 35S promoter. These plants showed
wild-type Arabidopsis phenotype, indicating that in spite of their sequence differences,
ZmTLK2 and TSL interact with the same substrates and regulatory partners and are
functionally equivalent.
© Copyright by Ethel Owusuwaa Owusu
June 28, 2004
All Rights Reserved
Characterization of the Zea mays ssp. mays TOUSLED-Like Kinases
by
Ethel Owusuwaa Owusu
A DISSERTATION
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented June 28, 2004
Commencement June 2005
Doctor of Philosophy dissertation of Ethel Owusuwaa Owusu
presented on June 28, 2004.
APPROVED:
7)
Redacted for privacy
Major
and Biophysics
Redacted for privacy
Chair of the Department of Biochemistry and Biophysics
Redacted for privacy
Dean of the Gra'duate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
I
Redacted for privacy
Ethel Owusuwaa Owusu, Author
ACKNOWLEDGEMENTS
I would like to thank my major professor, Carol Rivin. Her expertise, help and
support which were certainly invaluable to the success of this research are very much
appreciated. I am also grateful to members of the Rivin Laboratory, past and present
for sharing these many years with me. I am indeed grateful to members of the Tri-Lab
group (Rivin, Lomax and Fowler) for their help and support, particularly Maria
Ivanchenko for the many interesting scientific discussions with her.
My committee members were particularly invaluable to me, and I would like to
thank Barbara Taylor, Mary Slabaugh, Phil Mc Fadden, and Wil Gamble for their
help, support and suggestions for my research. I would also like to express my sincere
appreciation to John Fowler for his help and for the many discussions on this project.
I would also like to thank Dr. Judith Roe for providing seeds for the tsl-1 mutant
and for sharing unpublished data and stimulating scientific discussions on TOUSLED-
like kinases with me. In working with Arabidopsis thaliana, Jeff Leonard shared his
extensive expertise on transfonnation and genetics and provided information and
plasmids in addition to numerous scientific discussions for which I am grateful. I
would like to thank Isabel Vales for all the helpful discussions on mapping and the
oat-maize hybrid lines. I am also grateful to Indira Rajagopal for the many scientific
discussions and help with this project. Steven Meyer provided help with the
phylogenetic analysis. I am grateful to other colleagues of the
Biochemistry/Biophysics and Botany/Plant Pathology programs for their friendship
over the years especially those colleagues who provided helpful discussions and
support during this research particularly, Laura Meek and Afua Nyarko.
I very much appreciate the love and support of my family and friends
throughout this degree program. I appreciate the entire Owusu family for encouraging
me onwards. I am grateful to my father, Mr. A.K. Owusu and my Mother for instilling
in me a love of learning and for the sciences in particular. I am grateful to my uncle,
Dr. A. A. Owusu for letting me know that I can do anything I set my mind to. I am
also grateful to my Uncle Stanley for being such a big support. I hope I live up to the
expectations you all have of me. I am grateful to my McLagan family for supporting
and cheering me onwards and upwards through this program of study especially
through these final stages. Their acceptance and welcome were invaluable. Thank
you, Dad, for constantly reminding me that your love for me doesn't change whether I
pass or not. I am deeply grateful to Julia Blackmore and Engelene Chrysostom for all
their help, support and prayers. Thanks Julia, for staying up with me those late nights.
I am also grateful to Tanya, Shawn, Cheryl and all my prayer warriors. The bowl
must have tipped over.
I would like to express my sincere appreciation for my husband, Bensa
Nukunya, for being supportive, loving and patient as I finished this program. Above
all, I give the glory to the Living God for in Him, through Him, and for Him are all
things.
TABLE OF CONTENTS
Introduction and Literature Review ........................................................................... 1
Chromatin assembly/remodeling and genetic regulation ....................................... 1
The Protein Kinase Superfamily ............................................................................ 7
TOUSLED-like Kinases are a conserved Protein Kinase subfamily ................... 12
Investigation of TSL function in Arabidopsis ...................................................... 13
MammalianTLKs ................................................................................................ 16
Drosophila melanoganster TLK .......................................................................... 20
Caenorhabditis elegans TLK ............................................................................... 23
Summary of TLK functions ................................................................................. 26
Mutants in Chromatin Remodeling Genes in Plants ............................................ 28
The TOUSLED-like Kinase Multigene Family of Maize .................................... 29
Materialsand Methods ............................................................................................. 36
Sequence of ZmTlk genes .................................................................................... 36
Library Screening for ZmTlk Genomic Clones ............................................... 36
RNA preparations and Northern Blot Analysis ................................................... 38
RT-PCR and 5' RLM RACE for ZmTlk eDNA Clones ...................................... 38
TLKsequence analyses ........................................................................................ 39
Expression Analysis of the ZmTlks ..................................................................... 46
Reverse-Transcriptase PCR for Expression Analysis ...................................... 46
Semi-Quantitative RT-PCR ................................................................................. 47
Determination of the Map Loci of the ZmTlk genes ........................................... 48
Maize TOUSLED-like Kinase Mutant Identification .......................................... 50
Functional Complementation of the tsl-1 phenotype ........................................... 51
Plant materials and Growth Condition ............................................................. 51
Selection of tsl-1/+ and tsl-1/tsl-1 plants ......................................................... 51
Transgenic Plasmid construction ..................................................................... 52
Agrobacterium tumefaciens Transformations .................................................. 53
Plant Transformations ...................................................................................... 54
Selection and Analysis of Transgenic Plants ................................................... 55
DNA and RNA Analyses of Transgenic Plants ............................................... 56
Phenotypic Analysis of Transgenic Plants ....................................................... 56
TABLE OF CONTENTS (Continued)
Pag
Results
58
The ZmTlk genes: Isolation and sequencing ....................................................... 58
The ZmTlk genes: Comparison of their DNA and Translation products ............. 66
The ZmTlk genes: Comparison of translation products to TSL
andTSL homologs .......................................................................................... 75
The ZmTlk genes are ancient orthologs ............................................................... 84
TLK genes in other grasses .................................................................................. 87
Analysis of the mRNA expression levels of the TLK genes
in various maize tissues ................................................................................... 96
ZmTlk expression is higher in dividing tissues ............................................... 96
ZmTlk expression correlates with S-phase and is highest
in endoreduplicating tissues ....................................................................... 101
Investigation of ZmTlk Function ....................................................................... 104
Characterization of TUSC-TLK lines ............................................................ 104
ZmTLK2 is a functional homolog of Arabidopsis TSL ................................. 107
35S::nTSL-cTLK Transformants ................................................................... 113
35S: :TLK2 Transformants ............................................................................. 118
.
Discussion .............................................................................................................. 124
The maize ZmTLK multigene family consists of two ancient classes .............. 124
Sequence and Phylogenetic analysis of the Plant and Animal TLK proteins.... 131
ZmTLK2 is functionally equivalent to the Arabidopsis TSL protein ................ 133
ZmTlk genes are differentially expressed .......................................................... 136
Conclusions and Recommendations ...................................................................... 140
Bibliography ........................................................................................................... 142
APPENDIX ............................................................................................................ 162
LIST OF FIGURES
Figure
Pag
1
Schematic representation of factors involved in chromatin assembly
during DNA replication in S-phase cells............................................................... 6
2
Characteristic 300-amino acid protein kinase catalytic domain ............................ 9
3
Summary of TLK activities .................................................................................. 27
4
Cladogram of grass family members of the Pooideae, Oryzoideae,
Chloridoideae, and Panicoideae clades (taken from Kellogg, 1998) ................... 31
5
Hypothesis for the existence of 3 Tousled-like kinase genes in
Zeamays ssp. mays .............................................................................................. 34
6
PCR primer positions ........................................................................................... 59
7
Comparison of the genomic structures of?1a,
a genomic clone of ZmTlk 1 to Tsl, the Arabidopsis Tousled gene...................... 61
8
Comparison of the genomic structure of ?4a,
a genomic clone of ZmTlk 2 to Tsl the Arabidopsis Tousled gene ....................... 62
9
Diagrammatic representation of a comparison of the sequences
of the B73 genomic clones of ZmTlkl, ZmTlk2 and ZmTlk3............................ 63
10 Comparison of the 16 exons of Arabidopsis tsl and maize ZmTlk 1
andZmTlk2 .......................................................................................................... 65
11 Alignment of the mRNA sequences ofZmTlkl and ZmTlk2 ............................. 67
12 Amino Acid sequence alignment of plant TOUSLED-like kinase proteins........ 72
13 Domain structures of the TLK proteins ............................................................... 78
14 Alignment of the kinase domains of 12 TLK proteins ......................................... 79
15 Phylogenetic Relationships of the TOUSLED-Like Kinase protein .................... 83
16 Mapping the ZmTlk chromosomal loci by PCR analysis
of the oat-maize hybrid lines ................................................................................ 86
LIST OF FIGURES (Continued)
Figure
17 PCR amplification of Tik genes (exons 12-14 and intervening introns)
fromZea species .................................................................................................. 90
18 Alignment of Tik intron 12 sequences from eleven different Poaceae species. .. 91
19 PCR amplification of Tlk genes from non-Zea Poaceae families ........................ 95
20 Tissue distribution of ZmTlk transcripts as analyzed by RT-PCR and
restriction enzyme digestion ............................................................................... 97
21 Calculating the mRNA transcript levels of ZmTLK genes in maize tissues ....... 98
22 The mRNA expression level of ZmTlkl and ZmTlk2/3 is higher
in dividing than in non-dividing maize tissues .................................................. 100
23 Difference in the expression levels of maize Tlk and cyclin B 1 genes
in dividing and expanding root and leaf segments ............................................. 102
24 Patterns of ZmTlk expression in developing endosperms ................................. 103
25 The putative maize tik mutant families .............................................................. 106
26 Transgenic constructs used for transforming Arabidopsis plants ...................... 110
27 An example of genotyping of transgenic lines by PCR analysis ....................... 111
28 Expression of Transgenes .................................................................................. 112
29 Phenotype of the tsl-1 mutation (floral) and complementation
of the mutation with the pFGCnTSL-cTLK construct ....................................... 115
30 Phenotype of the tsl-1 mutation (gynoecium) and complementation
of the mutation with the pFGCnTSL-cTLK construct....................................... 116
31 Phenotype of the tsl-1 mutation in Arabidopsis leaves and complementation
of the mutation by the pFGC nTSL-cTLK construct ......................................... 117
32 Phenotype of the tsl-1 mutation (floral) and complementation
of the mutation with the maize TLK 2 gene ...................................................... 119
LIST OF FIGURES (Continued)
Figure
33 Phenotype of the tsl-1 mutation (gynoecium) and complementation
of the mutation with the maize TLK 2 gene ...................................................... 120
34 Phenotype of the tsl-1 mutation in Arabidopsis leaves and complementation
of the mutation by the pFGCTLK construct...................................................... 121
35 A Consensus grass comparative map (taken from Gale and Devos 1998) ........ 130
LIST OF TABLES
Table
1
Oligonucleotide Primer sequences used in this study.......................................... 41
2
Accession Numbers of TOUSLED-like Kinases................................................. 45
3
Primer pairs used in determination of the chromosomal
location of ZmTlk genes ...................................................................................... 49
4
Primer pairs used in transgene analysis............................................................... 56
5
Relatedness of monocot TOUSLED-like kinase genes
at the amino acid level .......................................................................................... 76
6
Phenotype of tsl-1 mutants compared to wild-type
Arabidopsis thaliana plants............................................................................... 108
7
Arabidopsis Transgenic Lines used in this study ............................................... 109
8
Analysis of Transgenic Arabidopsis Ti Lines ................................................... 114
9
Analysis of Transgenic Arabidopsis T2 lines .................................................... 122
10
Organ Numbers in Transgenic Plants ................................................................ 123
LIST OF APPENDIX FIGURES
Appendix
Pge
A
Zea mays mays (W22) T1kA-TIkXYr/Sacl ZmTlkl product sequenced
withprimer T1kA ........................................................................................ 163
B
Zea mays mays (W22) TIkA-TIkXYr ZmTIk2J3 product sequenced
withprimer TIkA ........................................................................................ 164
C
Zea mays mays (S60) TIkXYr/Sacl ZmTlklproduct sequenced with
primerT1kA ................................................................................................ 165
D
Zea mays mays (S60) TIkA-T1kXYr ZmTlk2/3 product sequenced with
primerT1kA ................................................................................................ 166
E
Zea luxurians (2003-101) T1kA-TIkXYr/Sacl ZmTlkl-Iike product
sequenced with primer T1kA ...................................................................... 167
F
Zea luxurians (2003-101) TIkA-TIkXYr ZmTlk2/3-like product
sequenced with primer TIkA ...................................................................... 168
G
Zea diploperennis (2003-102) T1kA-T1kXYr/Sacl ZmTlkl-like
product sequenced with primer T1kA ......................................................... 169
H
Zea diploperennis (2003-102) T1kA-T1kXYr ZmTlk2/3-like product
sequenced with primer T1kA ...................................................................... 170
I
Zea diploperennis (2003-103) T1kA-T1kXYr/Sacl ZmTIkl-like
product sequenced with primer T1kA ......................................................... 171
J
Zea diploperennis (2003-103) T1kA-T1kXYr ZmTlk2/3-like product
sequenced with primer T1kA ...................................................................... 172
K
Zea mays huc/zectenagensis (2003-104) T1kA-T1kXYr/Sacl ZmTlkl-like
product sequenced with primer T1kA ......................................................... 173
L
Zea mays huchectenagensis (2003-104) T1kA-T1kXYr ZmTlk2/3-like
product sequenced with primer T1kA ......................................................... 174
M
Zea mays mexicana (2003-105) T1kA-TIkXYr/Sacl ZmTlkl-like
product sequenced with primer T1kA ......................................................... 175
LIST OF APPENDIX FIGURES (Continued)
Appendix
ig
N
Zea mays mexicana (2003-105) TIkA-TIkXYr ZmTlk2/3-like product
sequenced with primer TIkA ...................................................................... 176
0
Zea mays parviglumis (2003-107) TIkA-TIkXYr/Sac 1 ZmTlk 1-like
product sequenced with primer T1kA ......................................................... 177
P
Zea maysparviglumis (2003-107) T1kA-T1kXYr ZmTlk2/3-like product
sequenced with primer T1kA ...................................................................... 178
Q
Tripsacum dactyloides (2003-108) TIIA-T1kXYr/Sacl ZmTlkl-like
product sequenced with primer T1kA ......................................................... 179
R
Tripsacum dactyloides (2003-108) T1kA-T1kXYr ZmTlk2/3-like
product sequenced with primer T1kA ......................................................... 180
S
Sorghum bicolor T1kA-T1kXYr product sequenced with primer T1kA ..... 181
T
Hordeum vulgare T1kA-T1kXYr product sequenced with primer T1kA.... 182
Introduction and Literature Review
TOUSLED-like kinases have been shown to be involved in regulation of
chromatin assembly during development. The complex nature of the cell and its
activities demand that all cellular processes be actively monitored and precisely
controlled. This is in order that inter- and intracellular communication and complex
functions be coordinated for optimal growth and maintenance of the cell and of the
organism as a whole. The cell achieves control in several ways including through
accessibility of DNA template modulated by chromatin remodeling, specific
subcellular locations of proteins, mRNA transcript availability and modifications and
by modification of protein activity through various means including protein-protein
interactions and covalent modifications of individual proteins by various means such
as acetylation, myristiolation and reversible phosphorylation. Phosphorylation events,
mediated by protein kinases and phosphatases are involved in numerous signaling
cascades including defense mechanisms (Hardie, 1997), cell cycle control and
differentiation (Morgan, 1997).
Chromatin assembly/remodeling and genetic regulation
Every nuclear process that requires access to DNA functions in the context of
the chromatin. This DNA access is modulated by chromatin assembly/remodeling
factors. Chromatin assembly and remodeling is tightly linked to DNA replication
(Krude, 1999). Nucleosome assembly is a two step process initiated by the deposition
2
first of a (H3.H4) tetramer followed by the addition of two (H2A.H2B) dimers
(reviewed in Krude and Keller, 2001). Both the process and machinery required are
well conserved in both plant and animal kingdoms being essential for proper
nucleosome function (Kaufhian and Almouzni, 2000). Further compaction of the
nucleosomes into chromatin fiber is initiated by the addition of histone Hi between
the nucleosomes followed by the deposition of non-histone proteins. Compaction
level of the nucleosome and modifications of the chromatin varies depending on the
need for access to the DNA. Although the nucleotide sequence of DNA determines
the sequence of the gene products, it is apparent that gene regulation through
inheritance in eukaryotes is also affected by chromatin state. One way of modifying
the chromatin is through the covalent modification of the N-terminal lysine-rich tails
of the core histones. These histone modifications are known as the histone code
(Goodrich and Tweedie, 2002). The histone tails, extremely conserved between
species, can be modified by remodeling enzymes including histone acetyltransferases,
deacetylases, protein kinases, ubiquitination enzymes and methylases. The histone
code may have a role in the generation of chromatin higher order structure and also
result in the activation or repression of gene expression. These active/silenced
chromatin states marked by the histone modifications are epigeneticaily inherited
through cell division (reviewed in Goodrich and Tweedie, 2002).
Work in Drosophila has served to accentuate the role of chromatin remodeling
as the basis for the stable changes in gene expression during epigenesis. This has been
shown to be responsible for phenomena like gene silencing and co-suppression (Pal-
3
Bhadra et al., 1999). One well studied group of proteins with a role in imprinted X
chromosome inactivation in mammals, transgene silencing in flies, cell proliferation
regulation and cell fate determination are the POLYCOMB group (PcG) of genes
originally identified in Drosophila (Pal-Bhadra et al., 1999). PeG genes are required
to ensure that the off state is maintained in cell lineages where a PcG target was
initially repressed and therefore form a cellular memory system that maintains the
repressed state of homeotic gene expression. PcG proteins are members of large
multimeric protein complexes. One such complex isolated from Drosophila embryo
extracts is the POLYCOMB-REPRESSNE COMPLEX 1 (PRC1) and contains
POLYCOMB and at least three other PcG members (Shao et al., 1999).
In plants, epigenetic regulation of gene expression is wide-spread. PeG genes
have also been identified in plants for example, FERTILIZATION INDEPENDENT
ENDOSPERM, (ZmFIE1 and ZmFIE2) in maize (Springer et al., 2002 and
Danilevskaya et al., 2003) and MEDEA, FIE and FERTILIZATION INDEPENDENT
SEED2 (FIS2) in Arabidopsis (Kohier et al., 2003). MEDEA, FIB, and FIS2 regulate
seed development in Arabidopsis by controlling embryo and endosperm proliferation
through epigenetic regulation of gene expression (Kohier et al., 2003). While the
function of the ZmFIE genes have not yet been elucidated, their sequence similarity to
Arabidopsis FIE and their expression pattern in endosperm and other maize tissues
may indicate roles in maize endosperm development (Springer et al., 2002 and
Danilevskaya et al., 2003).
I
Other epigenetic phenomena seen in plants include paramutation and parent-oforigin, i.e., imprinting effects (Goodrich and Tweedie, 2002). These phenomena are
reversible. An example of epigenesis in the maize plant is the imprinting of the R
gene in the endosperm. The R gene is required for red anthocyanin pigmentation in
the aleurone layer of the endosperm. In the absence of R activity, kernels are yellow
due to carotenoid pigments. The regular R allele that produces red kernels is
designated R-r:std. The r-g allele of R produces kernels that lack anthocyanins. R-
r:std" has the exact same sequence as R-r:std however it is an imprinted allele of R-
r:std. Kernels derived from an R-r:std"/R-r:std" male X r-glr-g female are yellow and
from the reciprocal cross are red. The R-r:std" allele is epigenetically silenced so that
almost no expression is obtained when it is paternally transmitted owing to imprinting.
The imprinting is correlated with differential methylation and it was shown that the
maternally expressed allele is demethylated relative to the paternal allele in endosperm
(Alleman 2000).
A recent flurry of papers has served to illuminate the role of chromatin state
achieved by assembly/remodeling as the basis for the stable changes in gene
expression during epigenesis. Chromatin assembly during S phase is accomplished by
two different pathways (reviewed in Krude and Keller, 2001). The first pathway
known as the parental nucleosome transfer allows histones from parental DNA strands
to be recycled by direct transfer and deposition unto the newly replicated daughter
strand. This method contributes to the assembly of about half of the newly replicated
DNA into chromatin. The other half is assembled by a second pathway known as the
de novo
nucleosome assembly, and is mediated by chromatin assembly factors that can
act as histone chaperones. These histone chaperones target soluble histones to the
sites of assembly at DNA replication forks. Figure 1 illustrates this mode of assembly.
Identified histone chaperones include chromatin assembly factor 1 (CAP-I), anti-
silencing function protein 1 (Asfi) and nucleosome assembly proteins (NAP 1 and
NAP2).
CAP-i consists of three subunits, the human homologues being p 150, p60 and
p48. ASF1 and NAP 1/2 are single subunit proteins. CAP-i and ASF1 activities are
replication dependent while NAP 1/2 activities are not. Both ASPi and CAP-i bind to
acetylated histones H3 and H4. In addition to these factors, ATP-dependent
chromatin remodeling factors are required to generate the regularly spaced
nucleosome arrays (Mello and Almouzni, 2001).
During S phase, the parental nucleosomal array is disrupted by the passage of
replication forks, therefore to maintain and transmit the correct epigenetic state of the
cell, the chromatin must be as faithfully duplicated during DNA replication as the
DNA is. Chromatin assembly is therefore as well coordinated as the cell cycle and
responds to the integrating intra and intercellular signals. Chromatin assembly events
are also associated with DNA recombination, DNA repair and transcription (Kauthian
Almouzni, 1999). Chromatin assembly and DNA synthesis are linked by interactions
between CAF- 1 and the proliferating cell nuclear antigen, (PCNA), an association
important for the localization of the chromatin assembly factors to the site of DNA
synthesis (Shibahara and Stiliman, 1999).
newly
thesed
histones
4J
==
cvtoplaTn
CKII
NAP-I /2
RCAF
CAF-I
ACT
a
NAP-1/2
nucleus
I
I
cydin/Cdk2
I
+
rn
1
3
PCNA
asetnbIy mrirediate
newly asaemhled
nudeosonw
array of spad nucIeoeon
replicating DNA
Figure 1 Schematic representation of factors involved in chromatin assembly during
DNA replication in S-phase cells.
Nucleosomes and histones are shown in green, assembly factors in blue (NAP-1/2)
and purple (CAF- 1 and RCAF, i.e. Asfi with histones) and replication-associated
proteins (PCNA, proliferating nuclear cell antigen) in red. Assembly reactions are
represented by small open arrows and factors involved in positively regulating these
reactions are indicated by large filled arrows. From Krude and Keller, 2001.
7
CAF-1 and ASF1 have been shown to operate synergistically in chromatin
assembly during DNA replication and DNA damage repair-coupled assembly in yeast
(Tyler et al., 1999) flies (Umehara et al., 2002) and in humans (Mello 2002). ASF1
(Asfip) was first discovered in yeast, where overexpressing the protein causes
derepression of silencing (Li et al., 1997). It was subsequently shown to interact with
a histone acetyliransferase during chromatin silencing in yeast. ASF1 binds directly to
the histone regulatory proteins (Hirp) and interacts genetically with, PCNA (Sharp,
2001). ASF1 forms a complex with the yeast DNA damage checkpoint kinase Rad53
(Emili et al., 2001).
The chromatin assembly factors are evolutionarily conserved. For example
homologues of ASF1 have been identified and characterized in humans (Sillje' and
Nigg, 2001) and fruit flies (Umehara et
al.,
2002). Database searches indicate ASF1
proteins in a wide variety of organisms including maize and
Arabidopsis.
While these
proteins are all very similar, the yeast Asfip, has a stretch of acidic amino acids at the
C-terminal end that the other proteins do not have and may be indicative of a function
peculiar to yeast Asfip.
The Protein Kinase Superfamily
Eukaryotic protein kinases (PK) are a superfamily of proteins that
phosphorylate proteins causing activation or inactivation of their functions. PKs are
involved in the regulation of cellular activity being involved in various control
mechanisms. They regulate the activity of metabolic enzymes providing a direct
['I
means of controlling cellular activity. PKs also phosphorylate transcription factors
resulting in changes in gene expression. A major part of PK function in cellular
communication is through signaling cascades. PKs phosphorylate other kinases
creating signaling cascades that relay information both inter and intracellularly or
amplify signals. Protein kinases are also involved in direct cell-cell signaling
(Manning et
al., 2002).
PKs comprise 1.5 to 3% of all eukaryotic genes (Manning
et al.,
2002).
Enzymes belonging to the PK superfamily share a conserved catalytic domain known
as the kinase domain (Hanks et
al., 1988).
The catalytic domains of PKs are 250
300 amino acids in length with alternating regions of high and low conservation. The
conserved regions are referred to as subdomains. There are 12 identified subdomains
in a kinase domain as shown in Figure 2. Invariant or nearly invariant residues are
indicated for each subdomain. The structures of several PKs have been solved by Xray crystallography and invariably, these conserved residues are found to be important
for both the 3-dimensional structure and activity of the protein (Wei et
a!, 1994).
Kinases perform their function in a three-step process: The enzyme first binds
and orients the y -phosphate donor (usually ATP or GTP) as a complex with a divalent
cation usually Mg2+ or Mn2+, It then binds and orients the protein/peptide substrate.
Finally it transfers the v-phosphate to the acceptor residue (ser, thr, tyr, or his) of the
protein substrate. This conserved mode of action is the reason for the conservation of
PK sequence.
I
GxGxxGxV
AxK
E
KxxDFG
II
GxxxxxAPE
I
HRDLKXXN
I DxWxxG I
J
R
I
Figure 2
II
III
IV V Via
Vib
VII
VIII
IX
X
XI
Characteristic 300-amino acid protein kinase catalytic domain.
The 12 conserved subdomains are indicated by roman numerals after Hanks and Hunter, 1995, Hanks and
Quinn, 1991 and Wei, et al., 1994. Conserved sequences found in some subdomains are shown. Invariant
residues are indicated as bold uppercase letters, nearly invariant residues are indicated as uppercase letters and
intervening residues are indicated by 'x'. Adapted from Stone and Walker, 1995.
10
The PK superfamily is subdivided into three major families tyrosine (Tyr)
kinases, serine and/or threonine (Ser/Thr) kinases and histidine (His) kinases. There
are a few atypical PKs that do not fall into these three classes (Ryazanov, 2002).
Examples of Tyr kinases include the Src (sarcoma) tyrosine kinase. It was first found
as a viral gene responsible for oncogenic transformation in chickens. Currently, no Tyr
receptor kinases have been found in plants. His kinases are a class of eukaryotic
kinases related to the prokaryotic two-component histidine kinases and are found in
plants serving as receptor kinases, an example being the Arabidopsis ETR1 gene, a
receptor kinase involved in ethylene sensing (Chang et
al.,
1993). His kinases have
also been identified in animals an example being the pyruvate dehydrogenase kinase
(reviewed in Besant
et al., 2003).
Examples of Ser/Thr kinases are the cyclin-
dependent kinases (CDKs), involved in cell cycle regulation (Nurse, 1990).
Within each family there are several distinct groups classified based on
sequence similarity in the kinase domain. Group classification is further refined by
similarities in biological function and extra-catalytic domain sequences (Hanks and
Hunter, 1995 and Manning et
al., 2002). All
major kinase groups and most kinase
families are found in metazoans and many are also found in yeast (Manning et
al.,
2002). This reflects the breadth of conserved functions mediated by kinases and
allows cross-species analysis of function.
In addition to regulating protein activity, the PKs are themselves regulated by
other kinases. An example of a well conserved group of kinases known are the CDKs.
CDKs are a kinase family found conserved from yeast to man (Morgan, 1997). CDK
11
activity fluctuates in a regulated manner during the cell cycle based on a sophisticated
machinery integrating intra and extracellular signals that leads to modulation of
cellular proliferation with these developmental and environmental cues. The cell
cycle is regulated at multiple points but the major points of regulation are at the Gi IS
and G2IM boundaries. Entry into S from Gi is controlled by CDK-A and cyclin D
complexes while the G2/M transition is controlled by the level of CDK and cyclin B
complexes (Dewitte and Murray, 2003). While the CDKs are the core cell cycle
kinases, other kinases are important in their regulation and in the regulation of other
aspects of the cell cycle. In eukaryotes, phosphorylation and dephosphorylation of
Thrl4 and Tyrl5 of the catalytic subunits of CDKs regulate their activity and
determine the timing of G2 and mitosis. Phosphorylation of Tyrl5 which leads to
inactivation of CDK1 is mediated by the related kinases Weel and Miki (Lundgren,
1991) and Myti (Liu, 1997). Weel was first identified in Schizosaccaryomyces
pombe to cause a delay in mitosis by phosphorylating the M-phase promoting factor
on Tyrl5. Subsequently, Weel functional homologues have been found in many other
organisms including Xenopus (Mueller, 1995), humans, (Liu, 1997), Drosophila
(Campbell, 1995) and maize (Sun, 1999).
Many protein kinases are members of families of closely related genes. Gene
families arise through gene duplications and are maintained due to their ability to
fulfill one or more of the following needs. First, duplicated genes can provide a level
of control of expression that a single gene is unable to provide. Second, duplicated
genes may provide for redundancy of an essential function and third they may also
12
provide overlapping functions in addition to divergent functions (Lynch and Conery,
2000).
It has come to light that various kinases play multiple roles in the cell. For
example, Weel in addition to its role at the G2IM transition also functions in the Sphase phosphorylation of CDK2/cylin E, CDK2/cyclin A and M-phase CDK1/cyclin
B (Campbell et aL, 1995, and Sun et al., 1999). These activities have been shown for
both plants and animals. While the study of the cell cycle has been pioneered in
animals and yeast, components of the cell cycle kinases have been found in plants and
generally the mechanism of regulation of the cell cycle is more similar (though there
are additional levels of control in each) to that in animals than in yeast (Huntley,
1998).
TOUSLED-like Kinases are a conserved Protein Kinase
subfamily
The TOUSLED-like kinases (TLKs) are another group of conserved protein
kinases found in higher eukaryotic genomes but not in the ESTs of fungi or the
genomes of S. cerevisiae and Neurospora crassa, implicating it in some aspect of the
development of multicellular eukaryotes or a layer of control not present in unicellular
eukaryotes. TLKs get their name from the founding member, the product of the
TOUSLED gene in Arabidopsis thaliana. The TSL gene encodes a Ser/Thr kinase
that is localized to the nucleus (Roe et al., 1997a). TSL homologues have been
identified in maize (Helentjaris et al., 1995, Yoon 1997 and this study), mice (Don and
13
Shalom, 1999), humans (Silije' et al., 1999), Drosophila melanogaster (Carrera et al.,
2003) and Caenorhabditis elegans (Han et al., 2003).
TSL has a unique kinase domain located at the C-terminal and an N-terminal
regulatory domain containing functional subdomains of coiled-coil regions and
nuclear localization signals (NLS) as well as a glutamine-rich (Q-rich) region of
unknown function (Roe et al., 1997a). TSL homologs (TLKs) have been found to
share at least 50% homology (higher in plants) in the kinase domain with TSL and the
N-terminal regions have similar functional motifs (i.e. coiled-coil regions and NLSs)
as TSL. The Q-rich region is not found in all TLKs. TLKs can be classified as a
conserved class of Ser/Thr PKs based on sequence and protein structure similarities.
Investigation of TSL function in Arabidopsis
In Arabidopsis, loss of TSL function mutants (tsl-1, tsl-2 and tsl-3) indicate an
important role for TSL in proper floral and leaf development (Roe et al., 1993,
1 997b). tsl mutants display a pleiotropic phenotype. tsl mutants are delayed in
flowering by about a week as compared to wild-type plants. The number of rosette
leaves is slightly increased in tsl plants and the margins of these leaves show deeper
serrations than wild-type. tsl plants form more secondary axes off the main
inflorescence than their wild-type siblings, and cauline leaves curl up tightly around
the new secondary axis in contrast to wild-type cauline leaves which unfold and bend
away.
14
The tsl mutant phenotype is most dramatic in flowers. Wild-type Arabidopsis
flowers are arranged in a bilaterally symmetric helical phyllotaxy. They have four
sepals, four petals and six stamens and a bicarpellate gynoecium i.e. a total of 14
organs not counting the gynoecium. In contrast, tsl flowers lack the symmetric
appearance of wild-type flowers, having fewer than 14 floral organs even though all
organs are in the correct positions (Roe
et
al., 1993). tsl flowers therefore appear
tousled or untidy. In tsl flowers, alllany organ type may be affected and the loss of
organs is random. On any one mutant plant, each flower is different in regard to the
organ complements that are present. While the gynoecium is always present, the most
important phenotype in tsl plants, however, is the lack of a fused gynoecium and
consequently tsl plants are sterile. Detailed examination of gynoecium development
showed that while all tissue types were present, they were at reduced levels (Roe
et
al., 1997b).
Based on this mutant phenotype, it was postulated that TSL most likely
participated in determining the pattern of initiation of floral organ primordia from
floral meristems and also may function during growth and differentiation of both
leaves and floral organs.
Arabidopsis TSL is a nuclear protein that requires autophosphorylation or
transautophosphorylation and dimerizationloligomerization for catalytic activity (Roe
et al., 1 997a). TSL is expressed in all Arabidopsis tissues, with highest expression in
floral buds and lowest in stem tissue. It was previously thought that non-receptor
kinases do not require dimerization to be active or as part of their regulation but
15
increasingly, instances of non-receptor kinases dimerizing have been reported in the
literature. In some cases, dimerization is necessary for activation, as in the case of the
rotavirus NSP5 protein (Torres-Vega et al., 2000) NSP5 is a non-structural
phosphoprotein that has kinase capabilities. Multimerization via its C-terminal region
is necessary for the protein to autophosphorylate. In other cases, while dimerization
occurs, it is not required for catalytic activity as evidenced by the double stranded
RNA-dependent protein kinase (Wu et al., 1996).
Dr. J. Roe's group at Kansas State University found that while Arabidopsis TSL
mRNA and protein levels are slightly higher in rapidly dividing Arabidopsis
suspension culture cells, these levels could not be aligned with a specific phase of the
cell cycle since levels of both mRNA and protein were constant for all stages of the
cell cycle (Ehsan et al., 2004). However, it is particularly noteworthy that TSL kinase
activity was found to be higher in G2IM cells than in S phase cells. This finding is the
opposite of that found in mammalian and fly cell cycle phases.
With the TSL protein activity indicating a role for the kinase at G2/M, Dr. Roe's
group compared the expression pattern of an M-phase cyclin, cyclin B1;l (maximally
expressed at G2/M in synchronized Arabidopsis suspension cells) in Arabidopsis wild-
type and tsl mutant plants. In tsl mutant plants, expression of the cyclin B 1; 1 was
found to be above wild-type levels in tsl floral meristems (Ehsan et al., 2004). TSL
may therefore be involved in the repression of cyclin B 1; 1 or in regulating epigenetic
control of cyclin B 1; 1 expression.
16
Arabidopsis As fib was found to interact with TSL in a yeast-2-hybrid assay. In
a separate yeast-2-hybrid screen, a novel protein, TSL kinase interacting protein
(TKI1) was found to interact with TSL (Ehsan et al., 2004). TKI1 contains a
SANT/myb domain and has homology to the Drosophila POLYCOMB protein
cramped (CRM). Myb proteins bind DNA and are transcription factors (Martin and
Paz-Ares, 1997). SANT domains are found in protein complexes that regulate
transcription (Boyer et al., 2002). These complexes are involved in chromatin
remodeling by interacting with both the histone N-termini tails and histone
acetyltransferases and deacetylases (Boyer et al., 2002 and Yu et al., 2003). Asfib
and TKJ1 appear to interact with TSL differently. While TSL interaction with Asflb
is via its N-terminus, its interaction with TKI 1 is strictly at the kinase
domain and the
N-terminal regulatory domain is not involved in this interaction. These results suggest
that TSL may have more than one role during Arabidopsis development
and has a role
in chromatin assembly/remodeling or regulation.
Mammalian TLKs
In humans, two TLKs (H5TLK1 and HsTLK2) that share 84% similarity with
each other at the protein level were identified and analyzed by Dr. Niggs' group at the
University of Geneva, Switzerland. Research in mice also identified two TLKs
(MmTLK1 and MmTLK2) with high homology to each other and several alternatively
spliced variants (Don and Shalom, 1999). Both human TLKs are localized to the
nucleus. Expression patterns of the MmTLKs and HsTLKs are similar. Both sets are
found in all tissues examined, with MmTLK1 and HsTLK1 showing a higher level of
17
expression in testis tissues (Silije'
et
aL, 1999, Don and Shalom, 1999 and Nagase et
al., 1995). The HsTLKs are capable of autophosphorylation and
transautophosphorylation, but autophosphorylation is not required for catalytic activity
(Silije et
al.,
1999). Like TSL, the HsTLKs dimerize.
The major breakthrough to discovering TLK function occurred from work on
the HsTLKs. Based on the hypothesis that the phenotype observed in
is a result of impaired cell proliferation (Roe et
al.,
tsl mutant plants
1 997b), Sillj e and co-workers
examined the expression levels and pattern of kinase activities of the two HsTLKs
during the cell cycle in synchronized HeLa cells (Silije'
et
al., 1999). HsTLK protein
levels are fairly constant through out the cell cycle but show differences in levels
of
phosphorylation. They are most highly phosphorylated in M-phase. The activities of
both kinases increased as the cell entered S-phase and decreased
upon exit from S-
phase.
Aphidicolin induces a block to DNA replication and leads to an arrest at GuS.
In aphidicolin-arrested cells, the TLK activity was almost undetectable. When
aphidicolin was added to cells in mid-S phase, TLK activity dropped within 5 15
minutes whereas in control cells, TLK activity only decreased as cells exited S-phase.
Inhibition was not correlated with phosphorylation state of the proteins. Neither
transcriptional inhibitors administered at S phase nor replication inhibitors
administered at Gl phase inhibited HsTLK activity. DNA damaging agents including
mitomycin (DNA cross-linking), bleomycin (DNA strand breaks) and camptothecin
(topoisomerase I inhibitor) inhibited TLK activity if added to mid-S phase cells.
LI1
HsTLK activity is therefore linked to ongoing DNA replication and is cell cycle
regulated. Based on these results, proposed roles for TLK genes included functions in
DNA replication and chromatin assembly and br remodeling.
In an investigation aimed at elucidating the regulation of TLK gene activity in
response to DNA damage and inhibition of replication, Niggs' group found that
inactivation ofHsTLK1/2 in response to DNA damage and inhibitors of DNA
replication is dependent on intact checkpoints rather than inhibition of DNA synthesis
(Groth et
al.,
2003). This checkpoint regulation of HsTLK1/2 is achieved in two
ways. First, in response to double stranded breaks, ATM (ataxia telangiectasia
mutated) kinase activates Chkl (central DNA damage checkpoint protein kinase)
which then transiently down regulates HsTLK activity. However when replication is
blocked due to environmental stress or random errors occurring during cell division, a
signaling pathway involving ATR (ataxia and Rad53 related) activates Chkl and down
regulates HsTLK activity. ATM, ATR and Chkl are involved in DNA damage
checkpoint regulation. ATM ATP-dependent phosphorylation of the histone variant
H2AX has been shown to be one of the first responses to DNA damage and is
necessary for efficient DNA repair (Bassing, 2002). These results suggest a plausible
link between TLK inhibition in response to DNA damage and DNA damage signaling
pathway to the regulation of chromatin assembly in mammalian cells.
Work on a splice variant of HsTLKs, indicate that HsTLKs are important for
stability and maintenance of the mammalian genome (Sunavala-Dossabhoy et
al.,
2003). Both mice and humans have two TLK genes in their genomes, however,
19
several alternatively spliced eDNA variants exist (Don and Shalom, 1999, Nagase et
al., 1995, Yamakawa et al., 1997, Li et al., 1999). One of the human variants,
HsTLK1B, phosphorylates histone H3 at ser 10 (Li etal., 1999). Phosphorylation of
histone H3 on Sen 0 has been associated with chromatin condensationlcompaction.
HsTLK1B when overexpressed in mammalian breast cell lines conferred
radioresistance to the cells and contributed to the development of radioresistant
carcinomas (Sunavala-Dossabhoy et
al.,
2003). HsTLK1B-KD, a dominant negative
mutant of HsTLK1B with no kinase activity, caused an increase in aneuploidy of
nuclei when overexpressed in a normal mouse cell line that normally shows diploid
chromosome state. The aneuploid number of chromosomes was highly variable and a
large number of these cells showed mitotic abnormalities. Typical mitotic defects
seen included a failure of a number of chromosomes to align properly at the
metaphase plate, failure of a number of chromosomes to attach to microtubules, the
occasional presence of two bipolar spindles with the centromeres of individual
chromosomes randomly attached to either of the spindle poles and the persistence of
microtubules bridges between two daughter cells after telophase and cytokinesis had
occurred. In addition, histone H3 phosphorylation was decreased in these cells. These
studies confirm that a link between HsTLK inhibition in response to DNA damage and
the DNA damage signaling pathway to the regulation of chromatin assembly in
mammalian cells.
20
Drosophila melanoganster TLK
Work in Drosophila embryos has yielded more evidence that TLKs cooperate
with ASF1 in cell cycle progression through the chromatin assembly and regulation
pathway (Carrera et al., 2003). DmTLK is a single copy gene in Drosophila.
DmTLK like TSL and HsTLK1/2 is localized in the nucleus and is widely expressed.
During embryo development, rapid DNA replication and chrornatin assembly occurs
during the early syncytial phase and DmTLK transcripts were observed to be highly
expressed at that stage. While expression of the DmTLK protein is enriched in
interphase nuclei, the proteins are not directly associated with the DNA but are rather
concentrated around the chromosome. Loss of function mutations in DmTLK (tlk14)
results in nuclear division arrest at interphase and homozygous individuals die as
larvae.
DmTLK is an essential gene in Drosophila development. Drosophila embryo
development occurs in 17 well coordinated stages known as Bownes stages
(http://sdb.bio.purdue.edu/flyfaimainlimages.htm). During stage one, the first two
cleavage divisions are completed. In stage two, cleavage divisions three to eight are
completed and the nuclei begin to move to the periphery by division five. By the end
of the eighth division, the majority of nuclei are evenly arranged on an ellipsoidal
surface about 35 .tm beneath the membrane of the egg. Some nuclei remain centrally
located while others drop out from the periphery and give rise to the yolk nuclei.
There is a doubling of the number of nuclei after each division; at the end of the eighth
21
mitosis, about 200 nuclei populate the periphery and about 50 presumptive yolk nuclei
are located in the center of the embryo.
In tlkAl4 mutant embryos at stage one, only a few had DNA and those with DNA
actually only had a few enlarged nuclei in which the DNA existed unstructured or as
network-like structures indicative of defects in chromatin structure. In tlk'14 stage two
embryos, nuclei divide and migrate to the periphery of the cell but they harbor defects
and lead to apoptosis. Nuclei defects included DNA bridges (resulting from
incomplete chromosome segregation) and nuclei were observed to be heterogeneous in
size. In wild-type embryos, early nuclear divisions are synchronized but in tlk14
mutants, these divisions were asynchronous such that nuclei in both anaphase and
interphase were obtained.
In tlk14 mutants, histone H3 phosphorylation levels decreased but embryos
induced to overexpress DmTLK did not show an increase in Histone H3
phosphorylation. This implies that DmTLK is not responsible for the pbosphorylation
of histone H3 in Drosophila however, the reduction in H3 phosphorylation levels may
be due to the reduction in number of mitotic nuclei. When mutants in which DmTLK
is overexpressed were analyzed, it was found that overexpression of DmTLK led to
embryo lethality.
The Drosophila embryo undergoes a specialized cell cycle composed of S and
M phases with no intervening phases. To determine the effect of DmTLK
overexpression on proliferating cells engaged in a regular cell cycle, mitotic
22
recombination experiments to generate homozygous DmTLK mutant (tlkAl4) cells
during imaginal discs development were performed. Carrera and co-workers found
that the tlk"4 imaginal discs initiated only a few small clones which did not continue
to divide but disappeared from the tissue while wild-type twin clones continued
normal growth (Carrera etal., 2003). This confirmed that loss of TLK activity
induces cell death.
In a separate experiment, imaginal discs engineered to be overexpressing
DmTLK were analyzed. DmTLK overexpressing imaginal discs, formed fewer cells
that were enlarged. These results showed that the DmTLK is necessary for nuclei to
progress through the cell cycle from S phase and to be able to segregate chromosomes
properly and these processes are dependent on DmTLK activity levels.
Drosophila
salivary glands undergo endoreduplicating cycles in which DNA is
replicated but daughter strands are not separated. This results in large salivary
chromosomes referred to as polytene chromosomes. Overexpression of DmTLK or
DmASF1 results in smaller salivary glands composed of fewer cells. Polytene
chromosome structures are disrupted so that the typical banding pattern of wild-type
salivary polytene chromosomes is lost (Carrera et
al.,
2003). Therefore
overexpression of DmTLK andlor DmASF1 disrupts endoreduplication. Coexpression of DmTLK and DmASF1 resulted in aggravated mutant phenotype such
that salivary glands are even smaller and are composed of only a few cells that show
less endoreduplicated DNA. During larval growth, cells formed during the embryonic
stages grow bigger but no new cells are formed. The defects described above for
23
DmTLK overexpressing mutants therefore suggest that DmTLK levels are both
important for embryonic development and regulation of endoreduplication.
In spite of these deformities, larvae overexpressing DmTLK and/or DmASF1,
under the control of the leaky heat-shock promoter at 30°C, developed into adults. The
resultant flies were mostly female and lacked some organs, specifically a number of
the large thoracic bristles. Carrera et
a!
also showed that DmTLK and DmASF1 form
a stable complex unlike the transient association reported for HsTLK and HsASF
interaction (Groth et
al.,
2003). The DmTLK and DmASFI interaction is dependent
on the phosphorylation state of DmTLK. DmASFI is bound to DmTLK when
DmTLK is hyperphosphorylated during S-phase which is also the time of highest
DmTLK activity. From the work of Carrera et
al
in Drosophila (Carrera et
al.,
2003),
it can be surmised that TLKs participate in chromatin assembly by regulation of
chromatin assembly/remodeling proteins through ASF 1. Their work also establishes a
link between cell cycle progression and chromatin assembly in addition to DNA
replication.
Caenorhabditis elegans TLK
Data from Arabidopsis, humans and Drosophila show that the classification of
TLK genes as a conserved group of ser/thr kinases based on sequence similarities is
supported by functional conservation. However, it appears that there may be some
TLK genes having more than one role in development.
24
This hypothesis is firmly supported by research out of Dr. J.M. Schumacher's
laboratory at the University of Texas. Dr. Schumacher's group worked on
characterizing the function of the single TLK (CeTLK) gene of the nematode C.
elegans
during embryogenesis (Han et al., 2003). Like other TLKs characterized to
date, CeTLK is widely expressed and is nuclear localized. Like HsTLK1/2 and
DmTLK, CeTLK protein is highly expressed in interphase nuclei. It is, however, also
highly expressed in prophase nuclei with background levels seen in other phases of the
cell cycle.
CeTLK is an essential gene. CeTLK mutants (fikRNAi) were generated by
RNA-mediated interference by injecting double stranded (ds) RNA or feeding young
adult hermaphrodites, bacteria expressing ds RNA corresponding to the CeTLK
cDNA. This resulted in fully penetrant embryonic lethality in the broodlprogeny of
treated animals. tlkRNAi embryos arrested development with about 100
undifferentiated cells which showed aberrant chromosome morphology. C.
elegans
embryos in which RNA polymerase III (RNAPII) activity is inhibited also arrest
development with about 100 undifferentiated cells (Powell-Coffinan et
aL, 1996).
This indicated a role for CeTLK in transcription. To verify this theory,
transgenes composed of the regulatory regions of RNAPII-dependent embryonically
expressed genes fused to a green fluorescent protein (GFP) reporter were transformed
into wild-type, RNAPII-RNAi and tlkRNAi embryos. It was discovered that while in
wild-type embryos expression of the GFP reporter was normal, it was non-existent in
RNAPII-RNAi and greatly diminished in the tlkRNAi embryos (Han et
al.,
2003). It
appears therefore that in C.
elegans,
the embryo lethal phenotype observed is due to
defects in embryo mRNA transcription. However, CeTLK also has a role in
chromatin assembly due to the chromosomal aberrations observed in the tlkRNAi
mutants. Dr. Schumacher's group observed a couple of other very interesting results.
First, CeTLK phosphorylation of embryonic histone H3 could not be shown. Second
protein modifications associated with transcription elongation were dramatically
reduced in tlkRNAi embryos.
Transcription of mRNA begins with assembly of a pre-initiation complex which
includes RNAPJI and a set of general transcription factors that establish the start site.
The transition from initiation to elongation is coordinated through phosphorylation of
the RNAPII C-terminal domain (CTD) which is based on the repeat YSPTSPS.
During initiation, the CTD is phosphorylated on serine 5, after which the RNAPII is
subjected to a checkpoint which establishes that the machinery for product elongation
is in place (Komamitsky et
al.,
2000). The CTD is then phosphorylated on serine 2, a
modification that predominates on elongating RNAPII molecules (Komarnitsky et
al.,
2000). In addition, chromatin modifications that may provide a transcription memory
also occur during this process. For example histone H3 tail is methylated on lysine 36
specifically during elongation (Gerber and Shilatiford, 2003). Chromatin remodeling
and/or modification are needed in order to allow transcription to start and proceed.
Loss of CeTLK activity by RNA1 led to a reduction of CTD serine 2
phosphorylation and also decreased levels of histone H3 lysine 36 methylation. These
results suggest that in the loss of CeTLK activity, transcription initiation occurs but
26
elongation is unable to proceed. It is possible that a checkpoint to clear initiation and
proceed to elongation is enforced in the absence of CeTLK. These results indicated a
role for CeTLK in transcription elongation in addition to a role in chromatin assembly.
Summary of TLK functions
It appears then that there are two separate roles for TLK genes (Figure 3); one in
cell cycle progression linked with chromatin assembly (Silije et al., 1999, Carrera
al.,
2003, Ehsan
et al.,
2004) and another in transcription elongation (Han
et
et al.,
2003). In view of the need for chromatin remodeling for transcriptional needs, these
two roles may be related, so that chromatin assembly accomplished in S phase
provides the basis for transcription elongation. It is also entirely likely that these two
roles are distinct; TLKs interact with histone chaperones, i.e., ASF1 to achieve
chromatin assembly and hence proper cell cycle progression and TLKs interact with
other proteins (e.g. TKJ1) to regulate transcription or remodel cbromatin for
transcription to progress or TLKs may be part of a checkpoint signaling pathway for
transcription. Such separable roles have been documented for other kinases. For
example the kinase CDK-7 is involved in cell cycle progression and is the kinase that
phosphorylates the CTD serine 5, the event necessary for transcription initiation in
yeast (Gerber and Shilatiford, 2003).
27
IIEI
p
od
IFfr!
suimt?
/
FstoreH3
A
cmnsPiL11we
/
Cell cyde
Epigic
stability)
Tuiscnkii
rqssion?
cntlDls
al., 2W2
Figure 3
Summary of TLK activities.
TLKs interact with and phosphorylate ASF 1. Altered ASF 1 phosphorylation could
affect chromatin assembly in S phase andlor during DNA repair. TLKs may also act
on chromatin through histone H3. TLKs may affect transcription through altered
chromatin states or through the putative transcriptional regulator, TKI1, and other
unidentified substrates. TLKs may have additional functions via as yet unidentified
substrates.
28
In general, chromatin biology and DNA metabolism are invariably linked and
several proteins previously thought to only function in chromatin remodeling have
been shown to have roles in other aspects of DNA chemistry and this may be the case
with the TOUSLED-like kinases. An example of proteins that have roles in both
chromatin remodeling and DNA chemistry is the TIP6O complex. TIP6O was first
discovered to be a histone acetyltransferase but has since been shown to be involved in
DNA repair and apoptosis pathways (Ikura et al., 2000).
Mutants in Chromatin Remodeling Genes in Plants
Genetic screens for developmental patterning mutants in plants have lead to the
identification of genes with either established or predicted roles in chromatin
remodeling. The FASCIATA mutations (Fasi and Fas2) in Arabidopsis display very
similar phenotypes with disturbed cellular and functional organization of the shoot
apical meristem (SAM) and root apical meristems (RAM) in addition to disturbed
gene expression in these tissues (Kaya et al., 2001). The phenotype is indicative of
possible roles for FAS 1/2 in the patterning and integrity of SAM and RAM and in
genomic stability. Cloning and sequencing of the responsible genes showed that FAS
1 and FAS 2 are subunits of the Arabidopsis CAF-1 homologue p150 and p60
respectively (Kaya et al., 2001). Kaya and co-workers concluded that CAF-1 may be
involved in the stable maintenance of gene expression states by ensuring proper
propagation of epigenetie states through DNA replication.
29
An Arabidopsis mutant with similar phenotype to TSL is the Splayed (syd)
mutant (Wagner and Meyerowitz, 2002). The mutated gene responsible for the syd
phenotype was recently cloned and sequenced and is an ATP-dependent chromatin
remodeling factor belonging to the SWIISNF class (Wagner and Meyerowitz, 2002).
This supports the hypothesis that TSL is involved in chromatin remodeling in
Arabidopsis. The fact that mutations in the single copy TLK genes of Drosophila and
C. elegans cause embryonic lethality whereas in Arabidopsis, tsl mutant plants are
able to grow to maturity may be due to the plasticity of plant cells. While cell fate in
animals is fixed during embryogenesis, plant cell fate is more determined by position
and information derived from neighboring cells. This may allow defects in chromatin
from TLK inactivation to be non-lethal. It is also possible that in Arabidopsis,
another protein is able to compensate, at least partially, for TSL function in chromatin
assembly or that the pathway in which TSL is involved has other regulators and hence
allows defects in TSL to be non-lethal.
The TOUSLED-like Kinase Multigene Family of Maize
Evolutionary conservation of the TLK proteins in plant and animal development
justifies using Arabidopsis TSL as a model to identify TLK genes and pathways in
maize. Previous work from Dr. Rivin's laboratory had isolated three TOUSLED-like
kinases in maize (ZmTLK) based upon hybridization to the Arabidopsis TSL catalytic
domain (Yoon 1997). Analysis of the sequences of the ZmTLK showed that they are
orthologues of TSL. Initial comparison of the maize TLKs (ZmTLK) showed that
they are very similar to one another and can be divided into two classes based upon
30
sequence homology. Dr. Helentjaris and co-workers mapped TOUSLED-like kinase
sequences in maize to chromosome loci 1L, 4c-L and 5S (Helentjaris, 1995).
The presence of three TLK genes in maize is most likely as a result of the
origins of maize. Maize, an ancient tetraploid, is a member of the highly diversified
grass (Poaceae/Gramineae) family. A cladogram showing the relatedness of the grass
family is shown in Figure 4. The grass family includes some 10,000 species and about
660 genera, including all the major cereals, maize, wheat, rice, barley and oats. It also
includes crops like sorghum, sugarcane and millet. Grass family members are highly
diversified in genome size, much of which is due to differences in amounts of
repetitive DNA present in each genome (SanMiguel et al., 1996). While both rice and
maize are diploids, with rice having 12 chromosomes (2n = 24) and maize having 10
chromosomes (2n
20), the rice genome is one of the smallest in the grass family
being 430 Mbp, while the maize genome is 2500 Mbp. While part of this large
difference in size is due to the presence of retrotransposon repetitive sequences, a
significant part is due to extensive duplications in maize as compared to rice. 72% of
single copy genes in rice are duplicated in maize (Aim and Tankley, 1993). Genetic
and molecular mapping studies in maize have shown that there are large chromosomal
portions that are duplicated between non-homologous chromosomes in the genome
(Helentjaris, 1988 and 1995). Chromosome blocks IL and 5S are one such
duplication. These extensive duplications in maize are due to genome-wide
duplications.
31
pg ONA/2C
0.5 -
EJ2.3
7.8 9.6 1 .5
13.3
7.0
Aegilops longissima
Aegilops sharonensis
Aegilops searsi
Aegilopsspeltoides
AegilQps tauschii
Triticum urartu
Triticum monococcum
Taeniatherum caput-med.
Secale cereale
Pooideae
Hordeum vulgare
Bromus spp.
Aira spp.
Co,ynephorus caulescens
Vahlodea atropurpurea
Deschampsia antarctica
Alopecurus utriculatus
Briza maxima
Desmazeria spp.
Festucaspp.
Lolium spp.
Milium vernale
Nardus stricta
Oryza sativa
Hygroryza aristata
Zizania aquatica
Eleusine floccifolia
Eleusine indica
Oropetiumthomaeum
Chloris gayana
Pennisetum glaucum
Pennisetum americanum
Vetiveria zizanioides
Sorghum bicolor
Tripsacum dactyloides
Zea luxurians
Zea diploperennis
-
3.5
Oryzoideae
Chloridoideae
I
I
I
Panucoideae
Zea mays
Figure 4 Cladogram of grass family members of the Pooideae, Oryzoideae,
Chloridoideae, and Panicoideae clades (taken from Kellogg, 1998).
32
Maize is in the grass tribe, Andropogoneae, which has a haploid or base
chromosome number of five; however, a few members of this tribe including maize
and sorghum have a base chromosome number of ten indicating a tetraploid origin for
maize (Gaut and Doebley, 1997). Based on this data, mapping and genetic studies and
sequence data analysis of duplicated genes in maize, other members of the Zea genus
and other grasses including sorghum and rice, modern maize is considered to have
arisen as result of a segmental allotetraploidization event (Gaut and Doebley, 1997).
Gaut and Doebley define segmental allotetraploids as genomes that have arisen from
the hybridization of two species with only partially differentiated chromosome sets.
That four chromosomes do not pair up during cell division is due to extensive
rearrangement in the maize genome. There are, therefore, a number of duplicated
genes in maize that have redundant functions. For example, the narrow sheath
duplicate genes (nsl and ns2) are found on duplicated chromosomal loci 2L and 4L
indicating they are ancestral duplicate genes (Scanlon et al., 2000). NS 1 and NS2
perform redundant functions during maize leaf development. Plants homozygous for
mutations in both ns genes fail to develop wild-type leaf tissue in a lateral domain that
includes the leaf margin. Plants homozygous for a mutation in either the nsl or ns2
gene have wild-type leaves. Other duplicated genes have been shown to have
divergent functions. For example the recently characterized maize fertilization
independent endosperm, ZmFie (ZmFiel and ZrnFie2) genes are members of the
POLYCOMB group and may have roles in gene repression (Springer et al., 2002).
However, they are differentially expressed. ZmFiel expression is limited to
developing kernels and is active exclusively in the endosperm, whereas ZmFie2 is
33
expressed throughout development in various plant tissues suggesting possible non-
redundant roles in maize development (Danilevskaya et al., 2003).
The maize TLK genes are duplicated genes and as such present an opportunity
to study the evolution of a multigenic family in maize. These genes may or may not
display functional conservation in spite of their sequence homology. It may also be
the case that the ZmTLK genes have overlapping functions and individual divergent
functions as a result of sequence differences in their non-translated regions. Figure 5
is a model showing three hypotheses to explain the presence of three TLK genes in the
maize genome. The first hypothesis is that each maize progenitor had one TLK gene
and the third gene has arisen through a recent duplication of one gene in maize. The
second hypothesis is that there were in fact two TLK genes per maize progenitor and
one of these genes has been lost via a deletion event leaving three TLK genes in
maize. A third possible hypothesis is that there was one TLK originally in each maize
progenitor, however before the hybridization event to generate maize, a single gene
duplication arose in one progenitor (progenitor B) leading to two TLK genes in
progenitor B in non-syntenous positions. Allotetraploidization therefore yielded three
TLK genes in maize.
34
Model I
Segmental
ITLK
TLKA
TLK B
Single gene
Al lotetraploidization (2 in Zea) Duplication
in Maize
II1
ZmTLK B2
Model 2
Segmental
2TLK
TLKAI
TLK
Allotetraploidization TLKB1
TLK B2
ZmTLK Al
Gene loss
ZmTLKB1
in Maize
ZmTLKB2
(4 in Zea)
LModel 3
1 TLK in progenitor A
Segmental
Single Gene duplication
in progenitor B, 2 TLKs
p
Allotetraploidization
TLK B2
(3 in Zea)
Figure 5
Hypothesis for the existence of 3 Tousled-like kinase genes in
Zea mays
ssp. mays.
In model I, ZmT1kA and one of the ZmT1kB genes will be in syntenous positions but
ZmT1kB1 and ZmT1kB2 will be most similar to each other. This model predicts two
Tlk genes in other Zea grasses.
In model II, all three ZmTlk genes may be in syntenous positions. This model also
predicts three Tlk genes in other Zea grasses.
In model III, T1kA and one of the T1kB genes will be in syntenous positions but T1kB 1
and T1kB2 will be most similar to each other. This model predicts three Tik genes in
other Zea grasses.
35
The TOUSLED-like kinases have been shown to be important genes in both
plant and animal development with roles in chromatin assembly, cell cycle
progression and transcription. To determine whether the ZmTLK genes are members
of the evolutionarily conserved TOUSLED-like kinases, this study aimed at
characterizing the ZmTLKs at the molecular and functional level. The goals of this
research are to determine the sequence similarity of the maize TLK genes, the origin
of three TLKs in maize and the function of the ZmTLK family.
36
Materials and Methods
Sequence of ZmTlk genes
Library Screening for ZmTlk Genomic Clones
To obtain full-length sequences of the ZmTlk genes, maize genomic and cDNA
libraries were screened using cDNA probes derived from partial sequences ofZmTlkl
and ZmTlk2.
Probes for Screening
Partial maize cDNA sequences of the ZmTlk genes were used to probe genomic
and cDNA libraries. Sequences for the 3' end of three TLK genes were previously
identified from a B73 maize genomic library (Yoon, 1997). Partial cDNAs
corresponding to two of the genes were used for additional library screening:
"5'cEAR" is a subclone of the cEAR eDNA described in Yoon, 1997. It contains
parts of two exons for the catalytic region of ZMTLK1. "pVW4" is a subclone of a
eDNA obtained from Pioneer Hi-Bred International that contains two exons of the
ZmTLK2 catalytic region. The two subclones were used as probes in library
screening experiments.
Genomic and cDNA Library screening
The 5'EAR probe was used to screen a B73 whole corn seedling genomic
library (Clontech Laboratories, Inc.) while pVW4 was used to screen a second
genomic (B73 immature ear) library from Pioneer Hi-Bred International, Inc., a B73
seedling leaf eDNA library obtained from Dr. Barkan (University of Oregon) and a
B73 apical meristem cDNA library that was kindly provided by Dr. Fowler (courtesy
of S. Hake and L. Smith, U.S. Department of Agriculture-Plant Gene Expression
Center, Albany, CA). Duplicate plaque lifts were transferred on to Magnagraph 0.45
tm nylon filters (MSI). Filters were prehybridized for 30 minutes to two hours at
65°C in a rotating hybridization oven (Robbins Scientific) after being baked at 80°C
for an hour. Lifts were then hybridized with 1.5 X 106 cpmlml 32P-dCTP labeled
random-primed DNA (Feinberg and Vogelstein, 1984) in 5 to 10 mL of 250 mM
NaH2PO4, 7% SDS. Blots were washed once with 0.5X SSC, 0.1% SDS and twice
with 0.2X SSC, 0.1% SDS. Washes were for 30 to 45 minutes each in approximately
lOOml of buffer. Positive clones were visualized by exposing filters to X-omat AR Xray film (Kodak) overnight to three days with an intensifying screen (Dupont) or to a
phosphoimager detection screen (Molecular Dynamics) overnight and analyzed using
the ImageQuant program (Molecular Dynamics).
DNA purification, analysis and sequencing
Once genomic clones were isolated from the library, the Wizard Lambda DNA
Purification kit was used to extract DNA from positive clones according to the
manufacturer's protocol (Promega). Clones were digested with restriction enzymes
and blotted on to nylon membranes (MSI). Resultant Southern blots were then baked
and hybridized as described for plaque lifts above. Hybridizing restriction fragments
were then subcloned into pBluescript II SK- (Stratagene) and sequenced. All
sequencing was done by Oregon State University's Central Services Laboratory, CSL.
RNA preparations and Northern Blot Analysis
Northern blot analysis was utilized in order to detennine the transcript size(s) of
the ZmTlk genes. Total RNA was extracted from various tissues including immature
ear by the TRIzol total RNA isolation reagent according to manufacturer's instructions
(Invitrogen). Poly A RNA was prepared using the Poly ATtract IV System
(Promega). RNA was quantified using an Ultrospec 2000 spectrophotometer
(Pharmacia Biotech) at 260 nm. Electrophoretic separation of total or poiy A RNA
was performed on 1.2% agarose with 2.2M formaldehyde gels in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Sambrook et al., 1989). Gels were blotted and
probed with 32P-labeled fragments of the appropriate ZmTlk cDNA as described for
DNA gels.
RT-PCR and 5' RLM RACE for ZmTlk cDNA Clones
Reverse-transcriptase polymerase chain reaction (RT-PCR) and 5'RNA Ligase-
Mediated Rapid Amplification ofeDNA Ends (S'RLM RACE), with cDNA isolated
from root tips of maize inbred W22 as template, was performed to obtain the complete
coding and 5'untranslated sequences of the ZmTlk genes. Through BLAST searches
of the public databases with Arabidopsis TSL, a rice TLK bomolog, OsTLK was
found. The degenerate primers TLK os-Lr, TLK os-Pf and TLK nPr (Table 1), were
designed to amplify the regulatory region of OsTLK. Based on BLAST searches
using the rice TLK sequence, a maize sequence (A1185 7208) corresponding to part of
the first, all of the second and part of the third exons of the TLK non-catalytic region
sequence was found. Primer TLK nLr was designed from A1857208. ZmTlk genomic
and cDNA sequences were used to generate PCR primers (Table 1 and Figure 6).
Primers were synthesized by the central Services laboratory (CSL) and MWG Biotech,
Inc. The non-catalytic regions of ZmTlkl and ZmTlk2 Primer were cloned from W22
root seedlings via PCR using the primer pair nLr and TLK Srb.
Amplification
products obtained from this PCR were cloned into the pCR-Blunt Il-TOPO vector
(Invitrogen) and sequenced. Using primers based on the RT-PCR products, 5'RNA
ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) was performed
using the FirstChoicelM RLM-RACE Kit according to manufacturer's protocol
(Ambion) to isolate the 5'UTR of the ZmTlk genes.
TLK sequence analyses
ZmTlk sequences from clones obtained via library screening and PCR
amplification were analyzed using the Genetics Computer Group (Version 10)
software. All other TLK sequences except TaTLK (wheat) were downloaded from the
GenBank data base (www.ncbi.nlm.nih.gov) and compared to the ZmTlk genes using
BLAST. The TaTLK sequence was provided by Dr. Roe (Kansas State University,
personal communication). Proteins included in this analysis are shown in Table 2.
Protein sequences were aligned using ClustaiW program (Thompson et al., 1994).
Phylogenetic analysis was performed using parsimony and neighbor-joining methods
with the PAUP (V4b) program (Swofford, 1998). For the parsimony tree, bootstrap
resampling analyses with 1000 replicates was performed to assess the degree of
support for each branch on the tree obtained (Felsenstein, 1985) The trees were
40
displayed with the TreeView (Page, 1996) software program. Protein molecular
weights were calculated with ExPASy Prot Tool (Wilkens et al., 1998).
41
Table 1
Oligonucleotide Primer sequences used in this study.
Organism /gene
Zea mays
Primer name
ZmTlk A
Zea mays
ZmTlk Zr
Oligonucleotide sequence
5'GGT TCA TCC AAA CAT TGT CAG GCT
ATG GGA
5' TAT GTC AAG CAC CGG CGT ATC
AGAT
Zea mays/actin 1
ZmacT 1
Zea mays/actin 1
ZmacT 2
Zea mays/cyclin Bi
Zea mays/cyclin Bi
Zea mays/histone H3
Zea mqys/histone H3
Zea mays/Mutator
TIR
5'CycZmel
3'CycZmel
5'zmHH3
3'zmHH3
Agrobacterium
Rb-2
5' CAC TGG AAT GGT CAA GGC CGG
TTT C
5' AAC CGT GTG GCT CAC ACC ATC
ACCT
Mu 9294
T-DNA
thaliana
Zea maysIASFl-like
Zea mays/ASF 1-like
Zea mays
Zea mays
ZmAsf if
ZmAsf ir
Zea mays
Zm Tlk-Qr
Zea mays
Tik D
5' GCA CCT GCC TTC CTC TCA
5' ACT TCC TGT ACA CCG CCT TCT G
5' AAG CAG ACG GCG CGC AAG TC
5' GCG AGC TGG ATG TCC TTG GGC A
5'AGA GAA GCC AAC GCA (AT)CG CCT
C(CT)A TTT CGT C
5' CGT GAC TCC CTT AAT TCT CC
tumifaciens
Arabidopsis
Tsl-Re
ZmTlk4-Of
Zm Tlk-Qf
5' ATT TGA GAT GGC AGG TCT TG
5' GAA AGC TTA TAT ATG TTG GAT CA
5' GGT TCA GGA TGG AAG TTG ATA
5' GGC CGT GGC TCA AAG ACT GT
5' CTC AAA GA(AT) CA(AT) GA(CT) CAA
CAA A(AT) G
5' C(AT) T TTG TTG (AG)TC (AT)TG
(AT)TC TTT GAG
5' GGT GGT TTC TGA TTT GGC AGA CCT
TACC
Zea mays
Zea mays
Zea mays
Zea inays
Zea mays
ZmTlk3-Yhe
Zm T1k3-out
ZmTlkl-Pr
Zm Tlkl-Nr
Tik XYr
5' TTG ATA CAT GAA TGC AGC TCT T
5' TGT GAG CTA GTG CAA ATG ATC AC
5' ATG CAA CTT TGC CAC GTA ATG
5'AGG TTT TGT ATC TGT GAC GC
5' AGG TCA AAG CAT TCT GGA GGA
AGAT
Unless otherwise specified, primers were made to TOUSLED AND TOUSLED-like
kinase genes. Primer names ending in the number 1, 3 and 4 indicate source of gene
sequence is ZmTLK1, ZmTLK3 and ZmTLK2.
42
Zea mays
Tlk-Tf
Arabidopsis
Tsl.-15f
5' TCG TAA ATC ACT TAA GAAAAG
ACA AT
5' AGA ATG AUG TAA AAG ATC TGA
AA
Arabidopsis thaliana
Vector pFGC5941
Arabidopsis thaliana
Binary-F
TsI-3'utr2xba
Zea mays
ZmTlk4-3'utr2x
Zea mays
ZmTlk4-Vfsapl
Zea mays
ZmTlk4-3'Rb
Zea mays
Zm T1k4-ZRb
Arabidopsis thaliana
Tsl-5c
Arabidopsis thaliana
Tsl-3Rx
Arabidopsis thaliana
Zea mays
Tsl-FE
Tslf-O
5' GGT GAA TGC TCT CTC CAG GA
5' AAT TAC CAT GGG GCG CGC C
5' GCT CTA GAC AGA AAC TTT ATA
AGC TAT ACC A
5' GCT CTA GAT ATC CAT CCC CAT
CCA TAA TCT CA
5' CGG TAT GCT CIT CTA AAT CTT
CTT GGA AA
5' CGG GAT CCT CAG ICA AGG GTT
AAATG
5' CGG GAT CCT ACT TCT TTG CAT
ACG AGA G
Zm T1k4-5'Fcl
5' CCA TCG ATT AAA
GIG TTG A
5' TAG TCT AGA TCC
CIT TCT TC
5' CAA CAT CIA ATC
5'GGA TCG ATG AGG
GAG AAC TTT
AAC TTT CAG
TIC CTG AGA A
GGA ACT GGG
AAG GGA A
Zea mays
Zm T1k4-S'Fc2
5' GGATCG ATA GTC AAC AUG TAG
TAGA
Zea mays
Zm T1k4-LfNdel
5' GTT CGG TCC ATA TGT CGG GCT
CGT
Zea mays
Zm T1k4-
5' CGG GAT CCT CAG TCA AAG GUT
AAA TG
Zea mays
Zea mays
3'RbamHlNew
Tik eLfi
Ilk bNrev
Zea mays
Tik eZrev
5' GGA ATT CAT GGA CCA GGA GGA
5' CGG GAT CCT TGC TGC CTC GTT
TGAT
5' GGA ATT CCA CCG GCG TAT CAG
AT
Zea mays
Ilk Mr2-BgIII
5' GGA GAT CTC GGA CGC TTC AGG
GTA
Zea mays
Zea mays
Tik L-lbamHl
Ilk N-2bamHl
5' GGA TCC TTC ATG GAC CAG GA
5' GGA TCC TTG CTG CCT CUT TTG
AT________________________
Table 1
Oligonucleotide Primer sequences used in this study (continued).
43
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Tik Rr-2bamHl
Tik Nf-1
Tik Pr-2bamHl
Tlk n4Uf
Tik nLr
Zea mays
Tik nPr
Zea mays
Zea mays
Zea mays
Oiyza sativa
Mtklutr
Oiyza sativa
TLK nPr
Oyza sativa
TLKos-Lr
Zea mays
Zea inays
Zea mays
T1kRF4
TIk4SR
Tik Srb
Arabidopsis thaliana
Qbox
Zea mays
Tlk eRF
Zea mays
Tik rf
Zea mays
Tik rr
Zea mays
Tik Ar
Zea mays
Zea mays
Primer B
Tik ur
Zeamays
Tik uf
Zea mays
Zea mays
PRO IS
Intron Zi
Zea mays
Intron Z3
Mtk3utr
Mtk4utr
TLKos-Pf
5' GGA TCC TCT CTG CTT TCG ACA
5' CAT CAA ACG AGG CAG CAA AGA
5' GGA TCC TTT OCT ACC CTA TCA CT
5' ACA TAT TTC GTC CTG TAA GAC
5' AA(AG) CTC GC(AC) AA(AG) CTG
GAG GCA
5' GA(AG) GA(AG) CTT TC(GT) AAA
TCG (AC)G(GT) CA
5' AAT TTA CAA CAT ATA CAG
5' AGA TTT TAG GTC ATT GCT A
5' AAT GGG CCC GTT AAT GCA
5'A(Y)TC(Y)TG(N)CG(N)GA(Y)TT(N)GA
(N)AGC
5'GA(AG) GA (AG) CTT TC (GT) AAA
TCG (AC) G (GT) CA
5' C(NM)G(N)ATGGC(N)GG(N)AA(R)G
5' GTG TCG AAA GCA GAG AGG CA
5' TCT TTT ATT GCC TGT CCG TCT TC
5' TAA OAT CTT TTA TTG CCT GTC
CGTC
Table 1
5' CAR CAR CAR CAR CSR CAR CAR
CAR CA
5' GGA ATT CGT GTCGAA AGC AGA
GAG GCA
5' GGA AlT CAA ATG AAA ATC CTG
TCC GAl CTG CTG
5' GGA ATT CTG CCT TAT TCT CAT
TCT CGC TTC TTG CC
5' TCC CAT AGC CTG ACA ATG TTT
GGA TGA ACC
5' TCT GAT GGG TAT GTT TGC TG
5' GGG TGA TOG CAG TGA CGC TG
5' CAG CGT CAC TGC CAT CAC CC
5' ATT TGG GAA GCG GAC AGC CA
5' GTA AGG TGA GAT CAC ACT TTA
GCC AAG TTA AAC AAT G
5' ACT AAC CAG CAT CAA CGA ATT
CTC GGC GAC AAA TOG
Oligonucleotide Primer sequences used in this study (continued).
44
Zea mays
Intron Z4
Zea mays
TLK-XR4
Zea mays
TLK-XR1
5' GAA TTC TCA GCG CAA CTA GGA
GAC AGA ATG GTG C
5' CCT GAG AAG TGA GTT CCA TTC
cc
5' CCT GAG AAG TGA GCT CCA TCC
cc
Zea mays
Zea mays
TLK4
X Forward
Zea mays
TLK B
Zea mays
MTK XY
Zea mays
MTK4
Zea mays
TLK C
5' GAT CTC AGT CAA AGG GTA AAT
5' TCA CTT CTC AGG GAG CTG GAA
CCT ATT G
5' AGC TGG CTT TGA AGG GAA CTC
CAC TCT
5' TCT TCC TCC AGA ATG CTT TGA
ccl
5' RACE Inner
Primer
5'RACE Outer
Primer
Table 1
5' AAG GAG ATC TCA GTC AAA GGG
TAA ATG G
5' AAT GCA CGG AGA GTG GAG TTC
CCT TCA A
5' CGC GGA TCC GAA CAC TGC GTTT
GCT GGC TTT GAT G
5' GCT GAT GGC GAT GAA TGA ACA
CTG
Oligonucleotide Primer sequences used in this study (continued).
45
Table 2
Accession Numbers of TOUSLED-like Kinases.
Organism
Arabidopsis
thaliana
Zea mays
Common
Name
Thale
cress
maize
Zea mays
maize
Zea mays
maize
Oiyza sativa
rice
Triticum aestivum
wheat
Homo sapiens
humans
Homo sapiens
humans
Mus musculus
mouse
Mus musculus
mouse
Drosophila
melanogaster
Caenorhabditis
elegans
Anopheles gambiae
fruitfly
Worm
nematode
mosquito
Protein Name
Abbreviation
used
TSL, AtTSL
GenBank
Accession Number
L23985
ZmTOUSLED
-like kinase 1
ZmTOUSLED
-like kinase 2
ZmTLK I
AY644701
ZmTLK 2
AY496080
ZmTOUSLED
-like kinase 3
OsTOUSLEDlike kinase
TaTOUSLEDlike kinase I
HsTOUSLEDlike kinase 1
HsTOUSLEDlike kinase 2
Mm
TOUSLEDlike kinase 1
Mm
TOUSLEDlike kinase 2
DmTOUSLED
-like kinase
CeTOUSLEDlike kinase
AgTOUS LEDlike kinase
ZmTLK 3
Not submitted
OsTLK
ACO9 1811
TaTLK 1
AK111604
Not submitted
FTsTLK 1
NM 012290
HsTLK 2
NM_006852
MmTLK1
AK029773
MmTLK2
NMO1 1903.1
DmTLK
AF181637
CeTLK
AY450852
AgTLK
XM3 11117
TOUSLED
For each organism the most recent full length clones deposited in GenBank as at May
04, 2004 are the ones used in this study. The sequence for Triticum aestivum was
provided by Dr. J. Roe, Kansas State University.
46
Expression Analysis of the ZmTIks
Reverse-Transcriptase PCR for Expression Analysis
In order to determine the tissue distribution of the ZmTLK genes, RT-PCR
using total RNA isolated from various maize tissues as templates were carried out.
Maize inbred W22 plants were grown in either the Botany and Plant Pathology field
nursery or greenhouse in Corvallis, OR. Samples collected included dividing and nondividing, vegetative, floral and inflorescence tissues. Tissues were frozen in liquid
nitrogen upon collection and then transferred to 80°C until used. Total RNA was
extracted from tissues by the TRIzol total RNA isolation reagent according to
manufacturer's instructions (Invitrogen). RNA quality was checked by: 1) running on
ethidium bromide stained agarose gels to determine size and to look for any
degradation of the rRNAs, and 2) testing for maize actin 1 RT-PCR products in cDNA
preparations. RNA was quantified using an Ultrospec 2000 spectrophotometer
(Pharmacia Biotech) at 260 nni. cDNA was produced from 2ug of total RNA at 42°C
using the SuperScript II Preamplification System (Invitrogen) and a poly-T primer.
Using the cDNA product as template, RT-PCR amplification was performed as
follows: 25uL reactions were run in IX Qiagen PCR buffer, 4mM MgC12, 0.1mM
dNTP mix, 0. 1mM of each primer with 0.4U Taq polymerase (Qiagen). In each
amplification, two sets of primers are included, (unless otherwise noted), one set of
maize ILK primers (ZmTLK A and ZmTLK Zr) that amplify both classes of ZmTLK
genes, and one set for maize actin 1 (ZmacTl and ZmacT2), as a positive and
quantitative control. In addition, the expression pattern of two maize cell cycle
components: the B 1-type cyclin, CycZmel, and histone 113 were analyzed. Analysis
47
of cyclin Bi expression was done using the primer pair, 5'CyZmel and 3'CycZmel.
For histone H3, the primer pair used were 5 'zmHH3 and 3 'zml{H3. These primers
were synthesized by MWG Biotech Inc and are listed in Table 1.
Semi-Quantitative RT-PCR
For semi-quantitative analysis, cDNA templates were serially diluted four-fold
into sterile ddH2O, stored at -20°C and used within a month of preparation.
Amplification was performed on a Stratagene® RoboCycler® Gradient 96 machine.
The amplification procedure included a one time 5 mm denaturation at 94°C, followed
by 35 cycles of 1mm each at 94°C, 60°C and 72°C and a final extension at 72°C for
5mm. The Taq DNA polymerase, PCR buffer and MgCl2 used were from Qiagen.
The same PCR reaction mixture as described in the reverse-transcriptase PCR for
expression analysis was used.
RT-PCR products were electrophoretically separated on ethidium bromide
stained 1.2% agarose gels and initially visualized under ultraviolet light with an EPI
Chemi II Darkroom camera from UVP Laboratory Products. Amplification products
were then visualized and quantified using the FMBi0 II fluorimager and software
(MiraBio). ZmTlk, cyclin B 1 and histone H3 quantitation values were divided by the
amount of actin 1 amplification in the specific reaction. These normalized values
were then plotted as a function of the dilution series. The slope value derived from the
linear regression analysis of a set of data was taken to be proportional to the transcript
amount of the gene plotted.
48
Determination of the Map Loci of the ZmTIk genes
Oat-maize addition lines were used to map the individual ZmTLK genes to
maize chromosome arms. Dr. Ronald L. Phillips (University of Minnesota) and Dr.
Riera-Lizarazu (Oregon State University) and co-workers have developed a complete
panel of oat-maize addition lines, each carrying one of the ten maize chromosomes
(Riera-Lizarazu
et al.,
1996 and Kynast et
al.,
2001). DNA and tissue material from
all ten oat-maize addition lines (OMA1.1 through OMA1O.1) and the oat and maize
parents, Sun II and Seneca 60, were kindly given to us by Dr. RL Phillips (University
of Minnesota) and Dr. Riera-Lizarazu (Oregon State University). DNA was isolated
according to Dellaporta (Dellaporta, 1994). The DNA concentration was determined
using the Ultrospec 2000 spectrophotometer (Pharmacia Biotech) at 260 nm. PCR
assays were used to detect the maize sequences in the OMA material. Primer pairs
used are shown in Table 3. PCR products obtained were subjected to diagnostic
restriction enzyme digests (Sac 1 site present in ZmTlkl but not ZmTlk2 or ZmTlk3
while there is an Eco Ri site in ZmTllc2 and ZmTlk3 but not ZmTlkl) to differentiate
between the ZmTlk genes since primer pairs used amplify products from both ZmTlk
classes.
Table 3
genes.
Primer pairs used in determination of the chromosomal location of ZmTlk
Forward Primer
Reverse Primer
Restriction Enzyme
Diagnostic Test
ZmTlk C
ZmTlk Zr
EcoRl site in ZmTlk2 and
ZmTlk3
ZmTlk A
ZmTlk B
Sad site in ZmTlkl
ZmTlk A
ZmTlk XYr
Sad site in ZmTlkl
ZmTlk Tf
ZmTlk N4Uf
Afihl site in ZmTlkl and
ZmTIk2,
ZmTlk3 sequence unknown
ZmTlk C
ZmTlk D
EcoRl site in ZmTlk2 and
ZmTlk3
ZmTlk 5'fC2
ZmTlk Mrb
BglII site in ZmTlkl and
ZmTlk2,
ZmTlk3 sequence unknown
nPr
ZmTlk Srb
BglII site in ZmTIkl and
ZmTlk2,
ZmTlk3 sequence unknown
ZmTlk Rf4
ZrnTlk Srb
BglII site in ZmTlkl and
ZmTlk2,
ZmTIk3 sequence unknown
ZmTlk Lf
ZmTlk Mrb
BglII site in ZmTlki and
ZmTlk2,
ZmTlk3 sequence unknown
ZmTlk Lf
ZmTlk Nr
BglII site in ZmTlkl and
ZmTlk2,
ZmTlk3 sequence unknown
Maize TOUSLED-like Kinase Mutant Identification
To investigate the function of the ZmTlk genes in maize, maize mutants for
ZmTlks were obtained and analyzed. A large collection of maize plants containing
Mutator (Mu) insertions throughout the genome has been developed at Pioneer HiBred
International. Based on PCR amplification using Mu and ZmTlk specific primers,
twenty-six families of the collection were obtained from Pioneer as lines that
putatively contained Mu insertions within the Tik genes. These were grown at the
OSU Botany Farm and leaf tissue from each plant was collected. DNA was prepared
from tissues collected by the Dellaporta method (Dellaporta, 1994) and screened for
Mu insertions by PCR amplification using one of the following primers ZmTlk A,
ZmTlk B, ZmTlk D, ZmTlk 4, ZmTlk Vfsapl and ZmTlk Xr in combination with Mu
9412, a degenerate primer that amplifies the terminal inverted repeats of several
Mutator transposable elements. SOul PCR reactions were run in 1X Hot Tub PCR
buffer, 1.5mM MgC12, 10% sucrose, 0.25mM dNTP mix with 4Ong of pooled DNA as
template. Amplification was done in a TwinBiock EasyCycler (Ericomp), with 10
mm at 94°C once, followed by 40 rounds of I mm at 94°C and 62°C, 2 mm at 72°C
and a final step at 72°C for 5mm. 3U of Hot Tub DNA Polymerase (Amersham life
Science) was added after the initial incubation at 94°C. A pre-amplified product from
Pioneer was re-amplified as a positive control in these PCR reactions. A no template
control was also included. lOul of each reaction was loaded unto 0.7% agarose gel in
Tris-borate buffer (Sambrook, Fritsh and Maniatis, 1989) and separated by
electrophoresis. The resultant gel was Southern blotted according to Sambrook, Fritsh
and Maniatis, 1989 and probed with cEAR and MTK4-2 as described under library
51
screening. MTK4-2 is a subclone of the
12th
intron of ZmTLK2. Positive pools were
re-screened as individuals following the same procedure.
Functional Complementation of the tsl-1 phenotype
To investigate the function of the ZmTlk genes, a complementation analysis of
the Arabidopsis TSL mutant, tsl-1, by ZmTlk2 was performed.
Plant materials and Growth Condition
Arabidopsis thaliana mutant line tsl-1 used in this work is in the Wassilewskija
background and was obtained from Dr. J. Roe (Kansas State University). tsl-1, a
recessive mutation in the TSL gene of Arabidopsis, has been described by Dr. Roe
(Roe et al., 1993) and is reviewed in the Introduction and Literature review chapter of
this work. Plants were germinated in growth chambers at 21°C under constant light.
After germination, they were moved to growth rooms at 21°C with 16 hours light
followed by 8 hours of darkness.
Selection of tsl-1I+ and tsl-1/tsl-1 plants
In order to select tsl-1 plants for transformation by maize Tik genes, the tsl-1
allele was selected for in Arabidopsis seedlings. The tsl-1 mutant allele was formed
by insertion of a 1-DNA carrying the kanamycin resistance gene in the tsl gene (Roe
et al., 1993). Six tsl-1 families were tested for tsl-1 segregation. Seeds were surfaced
sterilized first with 95% ethanol followed by a 50% solution of bleach and rinsed 5
times with sterile water. After the last wash, seeds were resuspended in a 0.1%
agarose solution. Surface-sterilized seeds were sown on kanamycin selection plates,
52
to select for the tsl-1 allele. The selective media contained O.5X MS salts (Murashige
and Skoog, 1962) from Invitrogen/Gibco, IX vitamin mix, 3% sucrose and 25mg/L
kanamycin. Plates were made with 8g agar/L. 50 seeds were sown per plate in a grid
format. Plated seeds were placed at 4°C for 24 to 48 hours to vernalize seeds and then
moved to a growth chamber for two weeks. Plants containing the tsl-1 allele were
then transplanted into soil.
Transgenic Plasmid construction
To obtain the plasmids needed to transform the tsl-1 plants for complementation
of the TSL mutation, ZmTLK2, TSL and a chimeric construct of the N-terminal
regulatory region of TSL coupled to the C-terminal catalytic region of ZmTLK2 were
cloned into the binary vector pFGC5941. All PCRs performed in this section were
done using Jnvitrogen's Platinum Pfx DNA polymerase system to ensure high fidelity
sequence. To make the 35S:TSL (pFGCTSL) and 35S:ZmTlk2 (pFGCTLK)
constructs, TSL and ZmTlk2 eDNA were modified via PCR amplification to have an
XbaI site at the 3' end of the cDNA. For pFGCTSL, primers TSL-5c and TSL3'utr2xba was used while the primer pair zmTLK4-5'Fc2 and zmTLK4-3'utr2x was
used for pFGCTLK. In each case a small section of the 5'UTR and 3'UTRs were
included. Using a BsaAl site in the 5'UTR of TSL and the XbaI restriction site
introduced via PCR, the eDNA for TSL was inserted into binary vector pFGC5941.
For pFGCTLK however, an AscI site in the 5'UTR of ZmTlk 2 and the XbaI
generated in the 3'UTR was used to move the ZmTlk2 cDNA into the pFGC5941
vector. To create the 35:TSL-TLK (pFGCnTSL-cTLK) construct, the C-terminal
53
catalytic domain of ZmTlk2 was obtained by PCR using primers zmTLK4-Vfsap 1 and
zmTLK4-3 'utr2x and ligated in-frame to the N-terminal regulatory domain of TSL
inserted in pLIT28 (New England Biolabs). The resultant hybrid construct of the Nterminal of TSL and the C-terminal of ZmTlk2 was then moved into pFGC5941 and
named pFGCnTSL-cTLK. The vector pFGC5941 used in this study has a kanamycin
resistance (kanR) gene for bacterial selection, a basta resistance (bar') gene for plant
selection, a CaMV 35S promoter to drive the expression of the target sequence and a
TMV omega leader sequence. The 1,352-bp ChsA intron (from the petunia Chalcone
synthase A gene) was replaced with the gene of interest. Plasmid pFGC5941 was a
kind gift from Dr. J Leonard, Oregon State University and all other pertinent
information for this plasmid can be found on the plant chromatin website
(www.chromdb.org).
Agrobacterium turn efaciens Transformations
To transform the Arabididopsis tsl-1 plants, recombinant Agrobacterium
tumefaciens carrying the constructs described in the previous section were generated.
The constructs pFGC5491 (negative control), pFGCTSL (jositive control) pFGCTLK
and pFGCnTSL-cTLK were introduced into Agrobacterium tumefaciens. The
Agrobacterium tumefaciens strain used was EHA1O5
(Kans, Carbs, Strpt)
and was
grown at 28°C. First, a 4 mL overnight culture of Agrobacterium in YEP media (lOg
yeast extract, lOg peptone and 5g NaCl) was used to inoculate a 100 ml culture. This
culture was allowed to grow for four more hours and then spun down at 6000rpm in a
J-21 Sorval centrifuge (Beckman Instruments) for 15 minutes at 4°C. Pellet was
54
resuspended in lOOmI of ice-cold 75mM CaCl2 and re-spun. After the second
centrifugation, the pellet was resuspended in I mL ofthe CaCl2 solution. The
Agrobacterium was then divided into 200 mL aliquots for transformation. To
transform the cells, 2 .tg of each DNA /construct was added to the cells and incubated
for 30 minutes on ice. They were then incubated in liquid nitrogen for 5 minutes after
which they were placed in a 37°C water-bath for five more minutes. 1 mL of YEP
media was added and cells were incubated at 28°C with shaking for two hours.
Transformed cells were then plated unto YEP/kanamycin plates and incubated at 28°C
for two to three days during which time the recombinant Agrobacterium grew. These
cells were then visible on the selection plate and were sterilely transferred into liquid
YEP/kanamycin media and allowed to grow overnight. To confirm the presence of
the original constructs that went into the Agrobacterium, DNA was isolated from the
Agrobacterium overnight cultures using the Qiagen DNA Miniprep kit and used to
transform E. coli DH5cx so that more DNA could be obtained for diagnostic restriction
enzyme digest and sequencing.
Plant Transformations
Arabidopsis plants mutant for TSL (both homozygous and heterozygous plants)
were transformed with the recombinant Agrobacterium using the floral-dip method
(Clough and Bent, 1998). Briefly, 5 mL of recombinant Agrobacterium cultures were
grown overnight and used to inoculate 200 mL of YEP/kanamycin media. The next
day the large cultures were spun down and the cells resuspended in 5% sucrose
solutions. Silwet L-77 (Lehle Seeds mc) was added to 0.02%. Flowers (open and un-
55
opened floral buds) and all aerial parts of Arabidopsis adult plants carrying the tsl-1
allele were immersed in the bacterium solution and swirled for about 10 seconds. Pots
were then laid out horizontally in a flat, covered with a plastic dome and kept in dim
light for 24 hours. The following day, domes were removed and pots set upright and
moved back into the growth room. Dipping was repeated after about seven days if
new buds were visible. For each construct, at least 10 pots of twelve to eighteen
plants each were dipped. Transformed plants (TO plants) were then allowed to set
seed (Ti seeds).
Selection and Analysis of Transgenic Plants
Ti seeds were vernalized at 4°C for two to three days, added tolOO mL of a
solution of 0.1% agarose in a squirt bottle and then planted in flats of insecticide
treated-soil such that seeds were fairly well spread out on the soil. Approximately
25,000 seeds (about 0.5g) were planted in each flat. Transformed plants (Ti plants)
were selected by irrigating seeds/seedlings with water containing the herbicide Basta
(PowerForce, active ingredient glufosinate ammonium, or Finale concentrate, active
ingredient glufosinate ammonium, Bayer CropScience) at a concentration of 40 mL of
herbicide concentrate to iO liters of water. After about two weeks, transformed plants
are apparent and growing well. Non-transformed plants do not survive. Transgenic
plants were then moved from the flat into individual pots for further investigation.
Selected Ti plants were allowed to self and the resultant T2 seeds were treated as
above and analyzed for segregation and heritability of the transgene.
56
DNA and RNA Analyses of Transgenic Plants
Genomic DNA was isolated from both TI and T2 plants using a miniprep
version of the Dellaporta protocol (Dellaporta 1994). Individual Ti and T2 plants
were tested for the presence of the transgene and the tsl-1 mutant allele using the
following primer pairs.
Table 4
Primer pairs used in transgene analysis.
Target Gene
Primer Pair
tsl-1 allele
RB2 and TSL RE
pFGCTSL
Zmllk Vfsapl and TSL RE
pFGCnTSL-cTLK
TSL 1Sf and ZmTlk Xr4
pFGCTLK
ZmTlk A and ZmTlk Zr
Total RNA was isolated from specific T2 plants with the TRJzol reagent
(Invitrogen). RNA was treated with amplification-grade DNAase (Invitrogen)
according to the manufacturer's instructions. First-strand cDNA was synthesized with
the SuperScript II reverse transcriptase system (Jnvitrogen) according to
manufacturer's protocol. This was followed by PCR as described under expression
analysis using the primer sets indicated above for pFGCTSL, pFGCTLK and
pFGCTSL-TLK.
Phenotypic Analysis of Transgenic Plants
Selected Ti and T2 plants were analyzed phenotypically. All whole plant
pictures were taken with a Nikon Coolpix 995 digital camera mounted on an inverted
57
tripod (Department of Botany and Plant Pathology). Images of flowers and floral
parts were taken using an Olympus XZX12 dissecting microscope connected to an
Olympus KT9086666 camera and were viewed and processed using the MagniFire SP
program from Optronics.
Results
The ZmTIk genes: Isolation and sequencing
To obtain sequences for the maize Tik genes, their genomic DNAs and cDNAs
were cloned. Previous RFLP mapping by Helentjaris and co-workers (Helentjaris
et
al., 1995) and work in our laboratory had previously identified three partial genomic
and two partial eDNA clones of the catalytic region of the TOUSLED-like kinases
(TLK) in maize (Yoon
et
al., 1997) defining three distinct Ilk genes in maize.
Pioneer Hi-Bred isolated and sent to us six clones of the partial maize TLK catalytic
region. One of the Pioneer clones, Pio4A, corresponding to most of the catalytic
region, was subcloned. The subclone, pVW4, included the first 3 exons of the
catalytic domain and was used to screen several libraries at high stringency. The
screened libraries were a B73 whole corn seedling genomic library (Clontech, Inc.), a
B73 immature ear genomic library (Pioneer Hi-Bred) and a maize shoot apical
meristem cDNA library (S. Hake andL. Smith, U.S. Department of Agriculture-Plant
Gene Expression Center, Albany, CA). Seven genomic and six cDNA clones were
obtained. Analysis of the PCR products indicated that all six eDNA clones were of
the catalytic regions of the maize TLK genes.
From a B73 inbred library, two unique TLK clones were obtained, A.Gla and
G4a representing ZmTlkl and ZmTlk2 respectively. No new genomic clones of
ZmTlk3 were obtained. The X clones of ZmTlkl and ZmTlk 2 had inserts of 11.5Kb
Regulatory Region
Nf
Lfnde
5'UTR
Of
'
1
2
3
-
4-4-
Lbl
Mrb Nrb
Figure 6
4
If
RI
nPr
-'
'
5
6
7
Vfsapl
A
Xf
C
3'UTR
8
4-
4-
Pr
Sr4
Srb
9
10
Uf
11
4Ar
12
4Xrl
Xr4
13
14
4XYr
15
16
4-
4-
B
Zr
Zrb
4-44
Diagram representations of the ZmTLK 2 protein regions and corresponding eDNA.
The relative positions of PCR primers used in this work are shown above and below the eDNA diagram.
Primer sequences are listed in Table 1. Numbers refer to the 16 exons.
D
and 13.1Kb, respectively and were therefore thought to contain complete genomic
sequences of ZmTlkl and ZmTlk2 respectively.
These clones were mapped by restriction enzyme analysis of their inserts and
hybridization to pVW4. Restriction fragments which hybridized to the pVW4 probe
were subcloned and sequenced (Figure 7 and Figure 8). Based on the original
restriction maps of these X clones and the sequenced fragments, the orientations of the
gene sequence within the clones were determined and the flanking regions were
subcloned into pBlueScript (pBS) and sequenced.
Both XG1a and ?G4a contained the complete TLK catalytic region (exons 11
16) and the last four exons (7 - 10) of the N-terminal regulatory region of TOUSLED-
like kinases in approximately 7 Kb of sequence (Figure 7 and Figure 8). Further
sequencing of the region upstream of exon 7 in the ?. clones did not match known
TLK sequences. For ?G4a (ZmTlk 2) 4.7Kb was subcloned and sequenced and for
XG1a (ZmTlk 1)1.5Kb was sequenced while there yet remained 4Kb to sequence.
Based on the sequence information and the fact that introns in maize can be large,
these sequences were assumed to be intronic sequence. This assumption was
reinforced when genomic PCR reactions that attempted to cross this intron were
unsuccessful.
61
Arabidopsis TsI Genomic
5UTRlIIIIl II HI IRIII3UT1
1
234 56
7
8
9 10 11
12
13
14 15 16
ZmTlk 1 Genomic Clone, Ala
p1 sp4(5 .2Kb)
plsp5 (8.8Kb)
plsbl9 (1.9Kb)
plsbe33 (3.3Kb)
Figure 7 Comparison of the genomic structures of Ala, a genomic clone of ZmTlk 1
to Tsl, the Arabidopsis Tousled gene.
The partial genomic sequence of ZmTlk 1 covering the catalytic region and the last 4
exons of the N-terminal regulatory region (exons 7 -16) was determined by
sequencing of X clone 1 a and its subclones. Shaded boxes represent exons, white
boxes represent intronic sequences, and solid lines below the structures represent the
regions included in sequenced strategic subclones.
62
Arabidopsis Tsl Genomic
III!! llIII3UT1
1
2
3
4
5
6
7
8
9 10 11
12
13
14 15 16
1'
ZmTlk 2 Genomic Clone, ?4a
KUO (0.6Kb)
pE4 (4Kb)
pES7-1
pSAL26
p4OEOl
(3.5Kb)
(2.6Kb)
(1.75Kb)
Figure 8 Comparison of the genomic structure of X4a, a genomic clone of Zm Tik 2
to Tsl the Arabidopsis Tousled gene.
The partial genomic sequence of Zm Tik 2 covering the catalytic region and the last 4
exons of the N-terminal regulatory region (exons 7 through 16) was determined by
sequencing of clone 4a and its subclones. Shaded boxes represent exons, white
boxes represent intronic sequences, and solid lines below the structures represent the
regions included in sequenced strategic subclones.
63
jjI
cEt4A
Regulatoty Region
JIH
-
I:.
--
vyvy yvy
R 88%
ZmTLK3
I
gemc
-75%
Figure 9 Diagrammatic representation of a comparison of the sequences of the B73
genomic clones of ZmTlkl, ZmTlk2 and ZmTlk3.
Colored boxes on top of triangles indicate intronic sequences. Relationships between
introns are indicated by a color code explained on the left-hand side of diagram.
Introns that are completely disparate in their sequences are indicated by different
colors. Boxes below the triangles indicate exon sequences. Arrows indicate that the
genomic clones extend beyond the regions shown. In ZmTlkl and ZmTlk2, there is
no TLK sequence homology in those regions. The white dashed box in the ZmTlk3
clone indicates what appears to be a deletion of part of exon 15, exon 14 and the
intervening intron.
64
In order to obtain the N-terminal regulatory region coding sequences for the
maize TLK genes, reverse-transcriptase PCR (RT-PCR) was used with the primer Tik
Srb in conjugation with the degenerate primers (nPr and nLr) made from the predicted
cDNA of the rice TLK gene, OsTLK, and a maize 274bp EST (GenBank #A1857208),
corresponding to the first three exons of OsTLK. The resulting PCR products, which
contained partial sequences of exon 1 through exon 8, were cloned into the TOPO
vector (Tnvitrogen) and sequenced (Figure 10).
The 5' untranslated region (UTR) and the rest of the first exon for ZmTIk2 was
obtained by performing 5'RNA ligase-mediated rapid amplification of cDNA ends
(5'RLM RACE) with primers Tik Mrb, Tlk Nr, 5'RACE outer and 5'RACE inner.
The 5' RACE primers were obtained from Ambion as part of the FirstChoice
RLM-
RACE kit. The complete coding sequence of ZmTlk2 was confirmed by RT-PCR
amplification of a full-length sequence ZmTlk2 (pTLK4) from W22 seedling root
cDNA (Figure 10) and deposited in GenBank with an accession number of
AY496080.
The partial coding sequence of ZmTlkl was confirmed by RT-PCR
amplification of the ZmTlkl mRNA from maize seedling roots (primers Lib and Zrb,
product W2-7). This sequence has been deposited in GenBank with an accession
number of AY644701. To obtain the 5' sequences of ZmTlk 1, 5'RLM-RACE was
attempted but the resulting sequences could not be unambiguously assigned to
ZmTlkl.
65
Arabidopsis TsI eDNA
1
23
4 567 8
9 10 11
13 14 15 16
12
5'UTR
3'UTR
rTrrr
I
ZmTlk 1 cDNA
Sph
5"EAR
cEAR
LS2O
W2-7
Gcontig 1
ZmTlk 2 cDNA
A4
pVW4
LS4
TOPO5Mr
pTLK4
Figure 10 Comparison of the 16 exons of Arabidopsis
and ZmTlk
tsl
and maize ZmTlk
2.
Green boxes represent the 6 exons of the sequence-conserved catalytic
regions. Blue boxes indicate the N-terminal structure-conserved
regulatory regions. Black lines represent strategic RT-PCR amplified
regions (cDNA). The purple line, Gcontigl, represents the 5'UTR and
first 2 exons of ZmTlkl predicted from maize genomic sequences
obtained from GenBank, accession numbers: CC675660, CC014013 and
CG382684. Solid green lines indicate the 5' and 3' untranslated regions.
1
Recent database searches have yielded maize genomic clones of ZmTlkl that contain
the 5'UTR and the first two exons of ZmTlkl, which was compiled and converted to
eDNA to give Gcontigl shown in Figure 10.
There axe sequences in GenBank databases that may correspond to the third
maize TLK gene, ZmTlk3. However, these sequences are fragmented and appear to
be error-prone. In conclusion, sequences for complete cDNAs of ZmTlkl and
ZmTlk2, partial genomic sequences for ZmTlkl and 2 (from exon 7 through the
3'UTR) and partial fragmented genomic sequences for ZmTlk3 were generated. In
this study, ZmTlkl and 2 were analyzed in depth.
The ZmTIk genes: Comparison of their DNA and Translation
products
Based on genomic sequence similarity among the three TLK genes of maize,
they can be subdivided into two classes with ZmTlkl as one class and ZmTlk2 and 3
in another class. Figure 10 shows schematic representations of the cDNAs of ZmTlk 1
and 2. The cDNA sequences ofZmTlkl and 2 are aligned and shown in Figure 11.
The sequences used for the alignment is from the end of the first intron through the
3'UTR of both genes. The maize TLK genes are quite similar to each other. For the
available sequence, gene structure is conserved in all three genes. For ZmTlkl and 2,
gene structure remains conserved across their entire sequence with exon/intron
junctions and exons sizes being very similar for both genes. The putative coding
regions of ZmTlkl and 2 are 84% identical at the DNA level and 91% identical at the
amino acid level.
67
ZmTlkl TTCATGGACCAGGAGGAGCTGCCTGAGACTTCCTCTTCCGATGACGATAA
ZmTlk2 TTCATGGACCAGGAGGAGCTGCCTGAGACTTCCTCTTCCGATGACGATAA
ZmT1 kl CTGTGAGGAGTTCTTGATACAAAAGAITACCCTGAAGCGTCCGAGATCTC
ZmT1 k2 CTGTGAGGAGTTTTTGATACAAAAGAATACCCTGAAGCGTCCGAGATCTC
ZmT1 ki. CAGATGGTGATAACATGCTTGCTCTTGGAAATTTTGAGGGTTCGGTAAAT
ZmTlk2 CAGATGGTGACAACATCCCTGCTCTTGGATTTTGAGGGTTCATCAAAC
ZmT1 ki GAGGCAGCAAGATTTTAGGCGTcACAGATACAAAACCTTCCTTGGACAA
ZmT1 k2 GAGGCAGCAAAGATTTTAGATGTCACGGATACAAGACCATCCCTGGACAA
ZmTlkl TTCAAGTAGGACAAGGCCGTGGAAGGGGTCGTGCTGGTACAGGCC
ZmTlk2 TTCAAATAGGAAACAAGGTCGTGGAAGGGGTCGTGCTGGTACAGGCC
ZmTlkl GAGGGCGTGGCTCAGGGCTGCTGATCACACGATTGACTTCAACTTCC
ZmTlk2 GAGGCCGTGGCTCAAGACTGTTGATCAACCACGACTGACTTTGATTTCC
ZmT1 ki TCAGCAGTTGTAACATGGCCiJCTAGATAATTAACCAACAAGGAACC
ZmT1 k2 GCAGCAGTTGTCAATGGTCP.CTAGATAMTTAACCAACAAGGAACC
ZmT1 ki CCGATCGAGTGTTCAACTGGGCCATGACGACAGAGCTGCTTTACAGGACG
ZmT1 k2 CCAATCGAGTGTTCAACTGGGCCATGACGACAGGGCTGCTTTACAGGAAG
ZmT1 ki AATTGTCAACATTACGTGGCAAGTTGCATTTTTGGAGGAAGAGCTTTCT
ZmTlk2 AATTGTCAATGTTACATGGCAGTTGCATTTTTGGAGGAGGAACTTTCT
ZmTlkl AATCGCATCAAGAAGCAACAAATTACCATGAACTTAGTGATAGGTTAGC
ZmTlk2 AAATCGCGTCAAGACGCACp&TTACCATCAACTGAGTGATAGGGTAGC
ZmT1 k 1 AAAGGAATTGAAGGATCTCAGATCATGACCAACAAATGAGATTAAAGC
ZmT1 k2 AAAGGAATTGAAGGATCTCApGATCATGACCAACAAATGAGATCTAAGC
ZmTlkl AAATGAJ\TCCTGTCCGATCTGCTGATAGCTGTGTCAAAAGCAGAGAGG
ZmTlk2 AAATGAAGGTGCTGTCTGATCTGCTGATAGCTGTGTCGAAAGCAGAGAGG
ZmT1 kl CAAGAAGCGAGAATGAGAATGGCAGGAATCTTTCAGACTTGGAAATGT
ZmT1 k2 CGAGCGCGGATGAGGATAAGGCAGGATCTTTCAGACTTGGGAATGT
ZmTlk]. TGCTGTTATGAGAGCTGGAACTATCATATCTGAAACTTGGGAAGACGGTC
ZmT1 k2 TGGTGTTATGAGAGCTGGAACTATCATATCTGAATTTGGGAAGACGGAC
Figure 11 Alignment of the mRNA sequences of ZmTlkl and ZmTlk2
The mRNA sequences of ZmTlkl and ZmTlk2 were aligned using ClustaiW. The
sequences used start from the end of the first exon and end in the 3'UTR of both
genes. Identical residues are in magneta. Similar residues are in blue. Distinct
residues are in black.
ZmTlkl AGGCAATAAAAGATCTTAATGCTCACCTGAATCTTTGCTGGAACTAAG
ZmTlk2 AGGCAATAAAAGATCTTAATTCTCACCTGAAATCTTTACTGGAAACTAAG
ZmT 1k 1 GAGACTATTGAAGGCATCGTAATCACTTAAGAAfiAGACAATCTGGTGA
ZmT1 k2 GAGACTATTGAAGGCATCGTAATCACTTAAGAAAGACAATCTG
A
ZmT1 ki CAAGGGTGATGGCAGTGATGCTGAGACTGGCATGTCTGAGGAGGACATCC
ZmT1 k2 CAAGGGTGATGGCAGTGACGCTGAGACTAGCATGTCCGAGGAGGACATTG
ZmT1 kl TCTTACAAGATGAAATATGTAAATCTCGGCTAATGAGTATAP.AACGGGAG
ZmTlk2 TCTTACAGGACGAAATATGTPAATCTCGTCTAACCAGTATCAAACGGGAG
ZmT1 kl GAAGAACAGTATATGAGAGAGAGAGATCGGTATGAGTTGGAAJAGGGGAG
ZmT1 k2 c3AAGAACAGTATATGAGAGAGAGAGATCGGTATGAGTTGGAAAAGGGGAG
ZmT1 ki GCTTATACGAGAGATGAAGCGTCTcAGAGATGPJGATGGCTCACGCTTTA
ZmT1 k2 GCTTATACGAGAGATGAGCGTCTCAGAGATGMGATGGCTCACGCTTTA
ZmTlkl ACAACTTCCAAATTCTTCACCATCGTTATGCTCTCTTAAATCTTCTTGGA
ZmTlk2 ACAACTTCCAAATTCTTCACCATCGTTATGCTCTGTTWTCTTCTTGGA
ZmT1 ki AAGGGAGGGTTTAGTGAGGTTTACAGGGCTTTTGATTTGGTGGAGTACAA
ZmT1 k2 AAGGGAGGGTTTAGTGAGGTTTACAAGGCTTTTGATTTGGTGGAGTACAA
ZmT1 ki ATATGTGGCATGCAGCTTCATGGATTGATGCTCAATGGAGTGAGGAGA
ZmT1 k2 GTATGTGGCATGCAGCTTCATGGATTGAACGCTCAATGGAGTGAGGAGA
ZmT1 kl AAAAACAGAGCTACATACGGCATGCGATTCGTGAATATAACATTCACAAA
ZmT1 k2 AAAAACAGAGCTACATACGTCATGCGATTCGTGAATATAACATTCACAAA
ZmT1 ki ACTTTGGTTCATCCACATTGTCAGGCTATGGGATATATTTGAGATTGA
ZmT1 k2 ACTTTGGTGCATCCAAACATTGTCAGGCTATGGGATATATTTGAGATTGA
ZmTlkl TCACATACATTCTGCACTGTCCTAGAATATTGCAGTGGCAAGGATCTTG
ZmT1 k2 TCACAATACATTCTGCACCGTCCTAGAATATTGCAGTGGCAAGGATCTTG
ZmT1 ki ATGCAGTCCTTWGCTACACCAATTCTTCCAGWAAGAAGGAAGGATC
ZmTlk2 ATGCAGTCCTTAAGCCACACCAATTCTTCCAGAAAAGAAGCAAGAATC
ZmT1 kl ATAATTGTTCAAATATTTCAGGGTCTGGTTTATCTAAACAAGAGGGGCCA
ZmT1 k2 ATAATTGTTCAAATATTTCAGGGCCTGGTTTATCTAAACAAGAGGGGCCA
ZmTlkl AAGATCATTCACTATGATCTGCCGGGCAATGTTCTCTTTGATGAGG
ZmT1 k2 AAAGATCATTCACTACGATCTAAACCTGGCAATGTTCTCTTTGATGAGG
Figure 11.
(continued).
Alignment of the mRNA sequences of ZmTlkl and ZmTlk2
ZmT1 ki TTGGTGTTGCAGTTACCGACTTTGGCCTCAGCAAGATAGTGGAGAT
ZmTlk2 TTGGTGTTGCAAAGTTACTGACTTTGGCCTCAGCGATTGTGGAGGAT
ZmTlkl GATGTTGGGTCTCAGGGGATGGAGCTCACTTCTCAGGGAGCTGGACCTA
ZmTlk2 GATGTTGGGTCTCAGGGAATGGAACTCACTTCTCAGGGAGCTGGACCTA
ZmTlkl TTGGTATCTTCCTCCAGTGCTTTGACCTGAGCAAAACACCATTTATTT
ZmTlk2 TTGGTATCTTCCTCCAGATGCTTTGACCTCAGCCACCATTTATTT
ZmTlkl CATCTAAGGTGGATGTTTGGTCAGCTGGTGTTGTTTTACCAGATGCTC
ZmTlk2 CATCTAGGTGGATGTTTGGTCAGCTGGTGTTGTTTTACCAATGCTT
ZmTlkl TTCGGAAAGCGTCCTTTTGGTCATGACCAGACCCAGGAGAGAJTACTTCG
ZmTlk2 TATGGAAGGCGTCCTTTTGGTCATGATCAGACTCAGGAGAGAATACTTCG
ZmT1 ki GGAAGATACGATTATCPTGCACGGAGAGTGGAGTTCCCTTCWGCCAG
ZmT1 k2 GGAAGATACAATTATCTGCACGGAGTGGAGTTCCCCTCAAGCC
ZmT1 ki CTGTATCAP.TGAGGCAJAGGATCTGATACGCCGGTGCTTGACATACAAC
ZmT1 k2 CTGTATCGTGAGGCGGGATCTGATACGCCGGTGCTTGACATACpJC
ZmTlkl CAGTCAGAGAGACCAGATGTACTCCATAGCTCP.GATCCTTATCTCTC
ZmTlk2 CAATCAGAGAGGCCAGATGTACTAACCATAACTCpGATCCTTATCTCTC
ZmTlkl ATACGCAGAGGTAGCCGCCATGCCGGAGCACAGGGTGTAACTGT
ZmTlk2 GTATGCAAAGAAGTAGACACTACCCCGGAGp.CATTTTG- -GCGATGGT
ZmTlkl ATATGTTGTAATTTTAGTCTTTCCCATcGCAGcAATGACCTGpTCCA
ZmTlk2 ATCTGCTCAGGCATTGAGATTATGGATGGGGATGGATAATCTC- -GTGGA
ZmTlkl GAAGCCATCAGTTCAACGGACAGCTTCTTTGTAGCGTAGAGGCAGG
ZmTlk2 AGAGCTAAGTATTApCA- -AGCTCAAGAAGACCACAAAGGGTGTATAA
ZmTlkl CATTGGTTTGACGGGGCATGCGTpCGTGATGGTTTCTTGGCTGGTGG
ZmTlk2 TTGTAAATTTTAGTCTGCCCTAT -TACGGCAATGACCTGAAATCCAGAAG
ZmTlkl ACTGTGGGCAGTGGTCCTGTTGATGGT -TGGTTTTTGTTTGCTCATACTG
ZmTlk2 GCATCAGTTCAGGCCAGCTTCTTTTGTAJCATAGAGAACATGCATTA
ZmTlkl TCGGTAPGGTCTGCC- - -AAATCAGA
ZmTlk2 ACGGGCCCATTTAACCCTTGACTGAGA
Figure 11.
Alignment of the mRNA sequences of ZmTlkl and ZmTlk2
(continued). The position of the stop codon of each gene is indicated by a solid black
line above the sequences.
,LI
ZmTlk 2 and 3 are 96% identical and 91% identical to ZmTlkl over the regions for
which sequence exists for all three genes (the 3 'UTR and the last two exons).
The 3 'UTR of all three genes are conserved. ZmTlk2 and 3 are 94% identical
and 75% identical to ZmTlkl in this region. The 5' UTR is also conserved and
ZmTlkl and 2 are 86% identical in this region. Intron size and homology varies from
intron to intron. ZmTlk 1 and 2 are 75% identical in intron 15, the last intron, and
69% identical in intron 9. However, in intron 8, they only share 20 base pairs in
common. The non-TOUSLED containing sequences in 2Gla and XG4a (which would
be intron 6) do not cross hybridize.
There are polymorphisms in these genes in various maize inbred lines. In B73, a
maize dent line, and W22, a different inbred, intron 12 of the ZmTlk2 gene hybridizes
to one TLK gene on a Southern whereas in Seneca 60 (S60), a sweet corn, ZmTlk2's
intron 12 hybridizes to two genes. Also a sequence which is present in all three
Tousled-like kinases genomic sequences of S60 appears to have been deleted in intron
15 of ZmTlk2 in B73.
The size of the sequenced ZmTlk2 mRNA is 2729 bp long and is expected to be
about 2800bp based on Northern blot analysis. The sequence for ZmTlk2 is believed
to be complete for several reasons. First, the 5'RACE method employed to obtain the
sequence is based on the recognition of 5 'capped mRNA (indicating full length
sequences). Secondly, there is an upstream stop codon before the first methionine,
which is in good Kozak context (Kozak, 1986). Thirdly, there is no homology with
71
other TLKs upstream of the starting methionine. ZmTIk2, therefore, codes for a
protein of 679 amino acids with a calculated molecular weight of 76.9 kDa. The
putative ZmTlkl sequence encodes a protein of 676 amino acids with a calculated
molecular weight of 76.5 kDa.
The predicted amino acid sequences of the corresponding translation products of
the ZmTLK proteins are shown in Figure 12, along with the sequences for Arabidopsis
TOUSLED protein (TSL, AtTSL) and the predicted rice (OsTik) and one of the wheat
(TaTlkl) TLK translation products.
72
OsTik MSGSSAAGEDIVQHLSSNSNPSS
- SKLAKLEARMAGKAAPVPSPPP
TaTiki MS
AAGEDIVQHLSSNSNPSS ----- SKLAKLEARMAGKAVSVPPPSP
ZmTlkl MSGSSAAGEDIVQHLSSNSNpSS
SKLAKLEARMAGKAVSAPS-SP
ZmTlk2 MSGSSAAGEDIVQHLSSNSNPSS
SKLAKLEARMAGKAVSAPS-SP
AtTSL
MS
OsTik
TaTiki
ZmTlkl
ZmTlk2
PHHLVVPS ------- APATTFMDQEE--LPESSSS--DDDNGEEFLIQKN
PHHPVVAP ------- ASAPTFMDQEE--LPESSSSS-DDDNGEEFLIQKN
PHHPMSAP --------- VISFMDQEE--LPETSSS--DDDNCEEFLIQKN
PHHPMAAPA ----- SAPAISFMDQEE--LPETSSS- -DDDNCEEFLIQKN
AtTSL
QQQQQQQQVSLWSSASAAvKVVTSTPPGLSETS I SDSDDENTGDFL IRAN
DDMVLHFSSNS SNQSDHSLPDKIAKLEARLTGKTPSSAKPPQ
OsTik I
TaTiki T
ZmTlkl T
ZmTlk2 T
RPR PDGDHGLAVGNFEGSANEAVK}.ISEVMDTRPS IDISN*KQGRG
RPR PDDDHGLALGNFEGSANAAAKVLDVMDTRLPSENPSRIKRQGRG
RPR PDGDNMLALGNFEGSVNEAAKILGVTDTKPSLDNSSPIKKQGRG
RPR PDGDNIPALGNFEGSsNEAAKILDVTDTRPSLDNSNRjKKQGRG
AtTSL
RQ
T
QESNNFSVVDHVEPQEAAYDGRKNDAESKTGLDVSKIKKQGRG
OsTik
RGGAGRGRGSKT-VDQTRATSTSSAVVANGRHDILTNMYQLNPYGDTL
TaTiki R3RGGTGRGRGSKT-VDQTRHTSASSVTVSNGQHDKLTNM ---------ZmTlkl R3RAGTGRGRGSRA-ADQTRLTSTSSAVVTNGQLDKLTNK ---------ZmTlk2 RIRAGTGRGRGSKT-VDQPRLTLISAAVVTNGQLDKLTNK
AtTSL
SS-TGRGRGSKTNNDVTKSQFvvAPvSAASQLDASDQK ----------
OsTik LLFAPSRLPLYAKEISYADQvyDGNLLHDDpSSSSKLLvSFHQESRSSAV
TaTiki------------------------------------------- DSRTSVL
ZmTlkl ------------------------------------------- EPRSSVQ
ZmTlk2 ------------------------------------------- EPQSSVQ
AtTSL------------------------------------------- DFRPDGQ
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
Figure 12
LGNDDKAALQEELSLLRGKVAILEEELSKSRQESTEYRQLSDRLAKELKD
PGNDDRTALHEELSLLRGKVAFLEEELNKSRQEVTEYHQLSDRLLAKELKD
LGHDDRAALQDELSTLRGKVAFLEEELSKSHQEATNYHELSDRLAKELKD
LGHDDRA.ALQEELSMLHGKVAFLEEELSKSRQDATNYHQLSDRVAKELKD
LRNGECSLQDEDLKSLRAKIANLEEELRKSRQDSSEYHHLVRNLENEVKD
Amino Acid sequence alignment of plant TOUSLED-like kinase proteins.
This alignment is based on sequences obtained on May 04, 2004. This alignment
shows the high homology between the plant TOUSLED-like kinases. Putative nuclear
localization signals, NLS, are indicated by solid bars and are boxed. The predicted
bipartite NLS of AtTSL is indicated by a dashed line. The first two NLS of AtTSL
have been shown to be functional (Roe et al., 1 997a) Abbreviations used are in Table
2. Globally conserved residues are in purple, identical residues are in red and similar
residues are in blue.
73
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
LKEQDQQKKSKQLKVLSDLLIAvSKAERQEARIRI KQESFRLGNVGVMRL
FKDQDQQKKSKQMKVLSDLLIAVSKAERQEARMKLKQESFRLGNIGVMRLKDHDQQMRLKQMKILSDLLIAVSKAERQEARMRIRQESFRLGNVAVMRLKDHDQQMRSKQMKVLSDLLIAVSKAERQEARMRIRQESFRLGNVGVMRLKDQEQQGKQKTTKVI SDLLI SVSKTERQEARTKVRNESLRLGSVGVLR-
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
-AGTVI SETWEDGQAI KDLNAHLKSLLETKEAVEHRK LKKRQSSDKGD
-AGTIISETWEDGQAIKDLNAHLKSLLETKETIEfHRK LKKRQSGDKGD
-AGTIISEIWEDGQAIKDLNSHLKSLLETKETIEjHRK_LKKRQS-DKGD
-TGTI IAETWEDGQMLKDLNAQLRQLLETKEAIERRKLLkKRQNGDKND
RAGTVISETWEDGQAIKDLNSHLKSLLETKETIHRK LKKRQSG-KGD
OsTik GSDAETSMSEEDVLLQDEICKSRLTS I KREEEQYLRERDRYELEKGRLIR
TaTiki GSDAETSMSEEDFLLQDEICKSRLTSIKREEEQYLRERDRYELEKGRLIR
ZrnTlkl GSDAETGMSEEDILLQDEICKSRLMSIKREEEQYMRERDRYELEKGRLIR
ZmTlk2 GSDAETSMSEEDIVLQDEICKSRLTSIKREEEQYMRERDRYELEKGRLIR
AtTSL GTDTESGAQEEDIIP-DEVYKSRLTSIKREEEAVLRERERYTLEKGLLMR
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
EM
EM
EM
EM
EM
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
CKLHGLNAQWSEEKKQSYIRHAI REYNIHKTLVHPNIVRLWDI FEIDHNT
CKLHGLNAQWSEEKKQSYIRHAIREYNIHKTLVHTNIVRLWDIFEIDHNT
CKLHGLNAQWSEEKKQSYIRHAIREYNIHKTLVHPNIVRLWDIFEIDHNT
CKLHGLNAQWSEEKKQSYIRRAIREYNIHKTLVHPNIVRLWDIFEIDHNT
CKLHGLNAQWSEEKKQSYIRHANRECEIHKSLVHHHIVRLWDKFHIDMHT
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
FCTVLEYCSGKDLDAVLKATPILPEKEARI I IVQIFQGLVYLNKRTQKII
FCTVLEYCSGIWLDAVLTPILPEKEARII IVQVFQGLVYLNKKAQKI I
FCTVLEYCSGKDLDAVLKATPILPEKEGRII IVQIFQGLVYLNKRGQKI I
FCTVLEYCSGKDLDAVLKATPILPEKEARI I IVQIFQGLVYLNKRGQKI I
FCTVLEYCSGKDLDAVLKATSNLPEKEARI I IVQIVQGLVYLNKKSQKI I
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
HYDLKPGNVLFDEVGVAKVTDFGLSKIVEDDVGSQGMELTSQGAGTYWYL
HYDLKPGNVLFDEVGVTKVTDFGLSKIVEDDVGSQGMELTSQGAGTYWYL
HYDLKPGNVLFDEVGVAKVTDFGLSKIVENDVGSQGMELTSQGAGTYWYL
HYDLKPGNVLFDEVGVAKVTDFGLSKIVEDDVGSQGMELTSQGAGTYWYL
HYDLKPGNVLFDEFGVAKVTDFGLS KIVEDNVGSQGMELTSQGAGTYWYL
RLjJEDGSRFNNFQILHNRYALLNLLGKGGFSEVYKAFDLVEYKYVA
RLR EDGSRFNNFQILHHRYALLNLLGKGGFSEVYKAFDLVEYKYVA
RLR EDGSRFNNFQILHHRYALLNLLGKGGFSEVYRAFDLVEYKYVA
RIJR EDGSRFNNFQILHHRYALLNLLGKGGFSEVYKAFDLVEYKYVA
RIR EDGSRFNHFPVLNSRYALLNLLGKGGFSEVYKAYDLVDHRYVA
Figure 12 Amino Acid sequence alignment of plant TOUSLED-like kinase proteins
(continued).
74
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
PPECFDLSKTPFI SSKVDVWSAGVMFYQMLFGRRPFGHDQTQERILREDT
PPECFDLSRTPFISSKVDVWSAGVMFYQMLYGRRPFGHDQTQERILREDT
PPECFDLSKTPFISSKVDVWSAGVMFYQMLFGKRPFGHDQTQERILREDT
PPECFDLSKTPFISSKVDVWSAGVMFYQMLYGRRPFGHDQTQERILREDT
PPECFELNKTPMI SSKVDVWSVGVLFYQMLFGKRPFGHDQSQERI LREDT
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
I
I
I
I
I
OsTik
TaTiki
ZmTlkl
ZmTlk2
AtTSL
KR
KR
KK
KK
INARRVEFP-SKPAVSNEAKELIRRCLTYNQAERPDVLTIAQEPYLSYA
INARKVEFP-SKPTVSNEAKELIRRCLTYNQSERPDVLSIAQDPYLSYA
INARRVEFP-SKPAVSNEAKDLIRRCLTYNQSERPDVLTIAQDPYLSYA
INARRVEFP-SKPTVSNEAKDL,IRRCLTYNQSERPDVLTITQDPYLSYA
IKAKKVEFPVTRPAISNEAKIJLIRRCLTYNQEDRPTJVLTMAQDPYLAYS
KR
Figure 12 Amino Acid sequence alignment of plant TOUSLED-like kinase proteins
(continued).
75
The ZmTlk genes: Comparison of translation products to TSL
and TSL homologs
To obtain insight into the evolutionary relationship between the maize TLK
genes, a sequence and phylogenetic analysis of TLK proteins from various species was
performed. The ZmTIk genes have high homology to Arabidopsis TSL and other
TOUSLED-like kinase genes (Figure 13 and Figure 14). At the amino acid level, the
ZrnTLK proteins are 84 % identical at the catalytic region to TOUSLED using the
Bestfit program of the GCG package. ZmTLK1 is 45% and ZmTLK2 is 49% identical
to TOUSLED in the N-terminal regulatory domain at the amino acid level.
Gene structure is extremely conserved between the currently known plant TLK
genes (TSL, ZmTlkl, 2 and 3, TaTLK and OsTLK). All the plant TLK genes have
exons of about the same size and iritrons are in exactly the same positions. Since both
TSL and OsTLK have 16 exons and 15 introns, the maize TLK genes are also likely to
have this structure. In maize and rice, the sizes of the introns appear to be conserved
in the C-terminal region. The genomic sequence for OsTik is 10270bp long. Based on
partial genomic sequences for ZmTlkl and ZmTlk2 in which intron 6 is bigger than 4
Kb whereas it is about 1 Kb in rice, the maize TLKs genomic sequences may be
bigger than the rice TLK gene.
76
The ZmTLK proteins are more closely related to each other than to rice or
wheat. However OsTLK is more closely related to ZmTlkl than to ZmTlk2 and the
reverse is true for TaTLK. All features of the ZmTlk genes are shared by OsTik and
TaTik, except for an insertion of 53 aa in the fourth exon of OsTik (Figure 12). Table
5 shows the relationship between the ZmTlk genes and OsTik and TaTik.
Table 5
level.
Relatedness of monocot TOUSLED-like kinase genes at the amino acid
ZmTIkl
ZmTIk2
OsTik
TaTiki
ZmTlkl
100
91%
79%
84%
ZmTlk2
91%
100
78%
82%
OsTik
79%
78%
100
74%
TaTiki
84%
82%
74%
100
77
TSL homologues have been identified in humans (Silije' et a!, 1999, Li et al.,
1999), mice (Shalom and Don, 1999), fruit fly (Carrera et al., 2003), nematode (Han et
al., 2003), and wheat (Dr. J. Roe, personal communication). A search of the GenBank
database revealed putative TLKs in several additional organisms but not in fungi
including Neurospora crassa and Saccharomyces cerevisiae. All the above mentioned
TLKs including complete cDNAs obtained from database searches for rice and
mosquito are included in the sequence analysis and are listed in Table 2.
The catalytic domain of all TLKs is conserved. Non-plant TLKs share a 43%
amino acid identity across the catalytic region and 25-27% identity with TSL when the
regulatory region is included. On the other hand plant TLKs are 83% identical in the
catalytic region, and 40-50% identical to TSL at the regulatory region. Figure 13 is a
schematic representation of the ILK proteins. Like TSL, all TLK including the maize
ones have their catalytic region at the C-terminal. Even though amino acid sequence
is not as conserved in the N-terminal regulatory region as it is in the C-terminal,
structural elements of the regulatory region are maintained in both kingdoms. In the
N-terminal domain, conserved features include nuclear localization signals and coiled-
coil regions. One feature which is not very well conserved however is a glutamine(Q) region. Only TSL, CeTik, and DmTlk have glutamine- (Q) rich regions in their
N-terminus. The maize, rice, wheat, mosquito, human and mouse TLKs do not have
any Q-rich regions. An alignment of the catalytic region of all currently known TLKs
(April, 2004) is presented in Figure 14.
78
ATTSL
688aa
78 KDa
ZmTLK 2 679aa
76.7 KDa
OsTLK
IUii II
733aa
82.7 KDa
1UISI II
TaTLK 677aa
76.7 KDa
HsTLK 2 749 aa
85.4 KDa
MmTLKI 718aa
81.8 KDa
CeTLK I 960aa
108.6KDa
DmTLK 1266a
l36.7KD1%%I
AgTLK 824aa
88.9KDa
Legend:
ii
-
rh
L.
I
Carboxy terminal kinase domain
-
INuclear Localization Signal (NLS). Pink refers to pat 4 NLS
(consists of four residues), black refers to pat 7 NLS (consists of
seven residues) and orange refers to bipartite NLS.
Coiled-Coil regions
Leucine zipper
Glutamine-rich (Q-rich) region
Figure 13 Domain structures of the TLK proteins.
Abbreviations are listed in Table 2. aa refers to amino acids and KDa is kilo Daltons.
79
I
Canonical
ZmTlkl
OsTik
ZmTlk2
TaTLK1
AtTSL
HsTlk2
MmTlk2
HsTlkl
MmTlkl
DmTlk
AgTlk
CeTik
gxGxxgxv
II
axK
RYALLNLLGKGGFSEVYRAFDLVEYKYVACKLHGLNAQWSEEKKQSYIRH
RYALLNLLGKGGFSEVYKMDLVEYKYVACKLHGLNAQWSEEKKQSYI RH
RYALLNLLGKGGFSEVYKAFDLVEYKYVACKLHGLNAQWSEEKKQSYIRH
RYALLNLLGKGGFSEVYKAFDLVEYKYVACKLHGLNAQWSEEKKQSYIRH
RYALLNLLGKGGFSEVYKAYDLVDHRYVACKLHGLNAQWSEEKKQSYIRH
RYLLLHLLGRGGFSEVYKAFDLTEQRYVAVKIHQLNKNWRDEKKENYHKH
RYLLLHLLGRGGFSEVYKAFDLTEQRYVAVKIHQLNKNWRDEKKENYHKH
RYLLLHLLGRGGFSEVYKAFDLYEQRYAAVKIHQLNKSWRDEKKENYHKH
RYLLLHLLGRGGFSEVYKAFDLYEQRYAAVKIHQLNKSWRDEKKENYHKH
RYLLLMLLGKGGFSEVHKkFDLKEQRYVACKVHQLNKDWKEDKKANY- - RYLLLMLLGKGGFSEVHKFDLKEQRYVACKVHQLNKDWKEDKKANYI KH
RYLMLNLLGKGGFSEVWKAFDIEENRYVACKIH-LDFYLKQNRSISE-
Consensus
Gr/kGGFSEV
AC/AK
ZmTlkl AIREYNIHKTLVHPNIVRLWDIFEIDHNTFCTVLEYCSGKDLDAVLKATP
OsTik AIREYNIHKTLVHPNIVRLWDI FEIDHNTFCTVLEYCSGKDLDAVLKATP
ZmTlk2 AIREYNIHKTLVHPNIVRLWDIFEIDHNTFCTVLEYCSGKDLDAVLKATP
TaTLK1
AtTSL
HsTlk2
MmTlk2
HsTlkl
MmTlkl
DmTlk
AgTlk
CeTik
Figure 14
AIREYNIHKTLVHTNIVRLWDIFEIDHNTFCTVLEYCSGLDAVLKATP
ANRECE IHKSLVHHHIVRLWDKFHIDMHTFCTVLEYCSGKDLDAVLKATS
ACREYRIHKELDHPRIVKLYDYFSLDTDSFCTVLEYCEGNDLDFYLKQHK
ACREYRIHKELDHPRIVKLYDYFSLDTDSFCTVLEYCEGNDLDFYLKQHK
ACREYRIHKELDHPRIVKLYDYFSLDTDTFCTVLEYCEGNDLDFYLKQHK
ACREYRIHKELDHPRIVKLYDYFSLDTDTFCTVLEYCEGNDLDFYLKQHK
ALREYNIHKALDHPRVVKLYDVFE IDANSFCTVLEYCDGHDLDFYLKQHK
Alignment of the kinase domains of 12 TLK proteins.
The abbreviated names used are defined in Table 3. Kinase subdomains are indicated
by roman numerals. Invariant residues are uppercase letters, nearly invariant letters
are lowercase and any intervening residues are designated as x. Kinase subdomains
are as defined in Hanks and Hunter, 1995 and Stone and Walker, 1995 and shown in
Figure 2.
E:II]
II
Canonical
E
VIb
hrDlkxxN
k
ZmTlkl
OsTik
ZmTlk2
TaTLK1
AtTSL
HsTlk2
MmTlk2
HsTlkl
MmTlkl
DmTlk
AgTlk
CeTik
ILPEKEGRI I IVQIFQGLVYLNKRGQKIIHYDLKPGNVLF]J- -EVGVAK
ILPEKEARII IVQIFQGLVYLNKRTQKIIHYDLKPGNVLFD- -EVGVAK
ILPEKEARII IVQIFQGLVYLNKRGQKIIHYDLKPGNVLFD- -EVGVAK
ILPEKEARIIIVQVFQGLVYLNKKAQKIIHYDLKPGNVLFD- -EVGVTK
NLPEKEARIIIVQIVQGLVYLNKKSQKIIHYDLKPGNVLFD- -EFGVAK
LMSEKEARSIIMQIVNALKYLNEIKPPIIHYDLKPGNILLVNGTACGEIK
LMSEKEARSIIMQIVNALKYLNEIKppIIHYDLKPGNILLVNGTACGEIK
LMSEKEARSIVMQIVNALRYLNEIKPPI IHYDLKPGNILLVDGTACGEIK
LMSEKEARSIVMQIVNALRyLNEIKppI IHYDLKPGNILLVDGTACGEIK
----- EARSIIIVIQVVSALKYLNEIKPPVIHYDLKPGNILLTEGNVCGEIK
TIPEKEARSI IMQVVSALKYLNEIKPPIIHYDLKPGNILLTEGNVCGEIK
- KEARSI IMQVVSALVYLNEKSTPIIHYDLKPANILLESGNTSGAIK
Consensus
E
HYDLKPg/aN
K
VII
Canonical xxDfg
ZmTlkl
OsTik
ZmTlk2
TaTLK1
AtTSL
HsTlk2
MmTlk2
HsTlkl
MmTlkl
DmTlk
A9T1k
CeTik
VTDFGLSKIVENDVGS- -QGMELTSQGAGTYWYLPPECFDLSKTP-FISS
VTDFGLSKIVEDDVGS- -QGMELTSQGAGTYWYLPPECFDLSKTP-FISS
VTDFGLSKIVEDDVGS- -QGMELTSQGAGTYWYLPPECF]JLSKTP-FISS
VTDFGLSKIVEDDVGS- -QGrIELTSQGAGTYWYLPPECFDLSRTP-FISS
VTDFGLSKIVEDNVGS -QGMELTSQGAGTYWYLPPECFELNKTP-MISS
ITDFGLSKIMDDDSYNSVDGMELTSQGAGTYWYLPPECFVVGKEPPKISN
ITDFGLSKIMDDDSYNSVDGMELTSQGAGTYWYLPPECFVVGKEPPKISN
ITDFGLSKIMDDDSYG-VDGMDLTSQGAGTYWYLPPECFVVGKEPPKISN
ITDFGLSKIMDDDSYG-VDGMDLTSQGAGTYWYLPPECFVVGKEPPKISN
ITDFGLSKVMDDENyNPDHGtIDLTSQGAGTyWyLPPECFVIJGKNPPKI SS
ITDFGLSKVMDEENYNPDHGMDLTSQGAGTYWYLPPECFVVGKNPPKI SS
ITDFGLSKIMEGESDDHDLGIELTSQFAGTYWYLPPETFIVP- -PPKITC
Consensus v/iTDFG
Figure 14
VIII
gxxxxxapE
Alignment
GTYWYLPPE
of the kinase domains of 12 TLK proteins (continued).
Canonical
Ix
DxWxxG
ZmTlkl
OsTik
ZmTlk2
TaTLK1
AtTSL
HsTlk2
MmTlk2
HsTlkl
MmTlkl
DmTlk
AgTlk
CeTik
KVDVWSAGVMFYQMLFGKRPFGHDQTQERILREDTI INARRVEFP-SKPA
KVDVWSAGVMFYQMLFGRRPFGHDQTQERILREDTI INARRVEFP- SKPA
KVDVWSAGVMFYQMLYGRRPFGHDQTQERILREDTI INARRVEFP- SKPT
KVDVWSAGVMFYQMLYGRRPFGHDQTQERILREDTI INARKVEFP-SKPT
KVDVWSVGVLFYQMLFGKRPFGHDQSQERILREDTI IKAKKVEFPVTRPA
KVDVWSVGVIFYQCLYGRKPFGHNQSQQDILQENTILKATEVQFP- PKPV
KVDVWSVGVIFYQCLYGRKPFGHNQSQQDILQENTILKATEVQFP- PKPV
KVDVWSVGVIFFQCLYGRKPFGHNQSQQDILQENTILKATEVQFP-VKPV
KVDVWSVGVIFFQCLYGRKPFGHNQSQQDILQENTILKATEVQFP-VKPV
KVDVWSVGVIFYQCLYGKKPFGHNQSQATILEENTILKATEVQFS -NKPT
KVDVWSVGVI FYQCLYGKKPFGHNQSQATILEENTILKATEVQFA-NKPT
KVDVWSIGVIFYQCIYGKKPFGNDLTQQKILEYNTI INAREVSFP-SKPQ
Consensus
DVWSa/v/iG
Canonical
ZmTlkl VSNEAKDLI:RCLTYNQSERPDVLTIAQDPYLSYAKR ----------OsTik VSNEAKELI RCLTYNQAERPDVLTIAQEPYLSY--AKR ----------ZmTlk2 VSNEAKDLIRCLTYNQSERpDVLTITQDPYLSY--AKK ----------TaTLK1 VSNEAKELIRCLTYNQSERpDvLSIAQDPYLSY--AKR ----------AtTSL ISNEAKDLIRCLTYNQEDRPDVLTMAQDPYLAY- -SKK ----------HsTlk2 VTPEAKAFIRCLAYRKEDRIDvQQLACDPYLLP- -HIRKSVSTSSPAGA
MmTlk2
HsTlkl
MmTlkl
DmTlk
AgTlk
CeTik
VTPEAKAFI RCLAYRKEDRIDVQQLACDPYLLP- -HIRKSVSTSSPAGA
VSSEAKAFIRRCLAYRKEDRFDVHQLANDPYLLP- -HMRRSNSSGNLHMA
VSSEAKAFIRRCLAYRKEDRFDVHQLANDPYLLP- -HMRRSNSSGNLHMS
VSNEAKSFIGCLAYRKEDRMDVFALARHEYIQP- PIPKHGRGSLNQQQ
VSNEAKSFI GCLAYRKEDRMDVFALAKHEYLQP- PVSKHNRSSNAQNA
VSSAAQDFI RCLQYRKEDRADVFELAKHELFRPRGAIRASVAGSVSSPS
Consensus
R
ZmTlkl-------------------------------------------OsTik
ZmTlk2-------------------------------------------TaTLK1 -------------------------------------------AtTSL
HsTlk2 AIASTS ------------- GASNNSSSN ---------------MmTlk2 AIASTS ------------- GASNNSSSN ---------------HsTlkl GLTASP ------------- TPPSSSIITY --------------MmTlkl GLTATP ------------- TPPSSSIITY --------------DmTlk QAQQQQQQQQQQQQQQSSTSQANSTGQTSFSAI-IMFGNMNQSSSS
AgTlk HAGGQN --------------- SSSTGTGA --------------CeTik IPRSPS --------------- VNREDDNM ---------------
Figure 14 Alignment of the kinase domains of 12 TLK proteins (continued).
The eleven subdomains typical of the catalytic site of SerlThr protein kinases
(Hanks et al., 1988 and Hanks and Quinn, 1991) are highly conserved. The kinase
subdomains are indicated by Roman numerals. All TLKs examined contain a
GXGGFS motif (where x is K for all TLKs except the mammalian ones in which it is
R) in the ATP-binding region (subdomain I) instead of the canonical GXGXXG
(Hanks etal., 1988 and Hanks and Quinn, 1991, Hanks and Hunter, 1995). In
subdomain II, the canonical AxK becomes ACK for all TLKs except the mammalian
ones in which it is AVK. All the twelve Tousled-like kinases studied for this analysis
also display a HYDLKPXN motif in subdomain Vib, the catalytic domain, instead of
HRDLKXXN. The absence of the RD motif in subdomain Vib indicates that TLKs
may not require phpsphorylation of a threonine residue in subdomain Vi for
activation. Subdomain VII becomes KVTDFG in plant TLKs and KITDFG in
metazoan TLKs conforming to KxxDFG. In subdomain VIII, the motif GxxxxxApE
becomes GTYWYLPPE in all TLKs while in subdomain IX DxWxxG is DVWSAG
in all TLKs of monocotyledonous origin, DVWSVG in Arabidopsis and the metazoan
TLKs except C. elegans in which it is DVWSIG.
The relationship between the TLKs is presented in Figure 15. Although the Nterminal sequences varies greatly between species, the cluster analysis produced the
same results whether the whole protein sequence or only the catalytic region
(corresponding to amino acids 319
relationship.
674 of ZmTlk2) was used to generate the
83
CeTik
DmTlk
Aglik
Mmllk2
Hsllk2
Hsllkl
Mmllkl
AtTSL
TaTLK1
ZmTIk2
ZmTIkl
OsTik
Figure 15 Phylogenetic Relationships of the TOUSLED-Like Kinase protein
The UPGMA method was used to analyze the catalytic region of TOUSLED-like
kinase proteins. This dendrogram shows that the TLK proteins from plant and animal
kingdoms form two well separated clades. This dendrogram is an indication of the
similarity between genes not species and was well supported. Abbreviations are listed
in Table 2.
The trees generated were unrooted and therefore only specifies the relationship
among the genes from different species, without identifying a common ancestor, or
evolutionary path. Two different methods were used to determine the phylogenetic
relationship of the TLKs. One used parsimony/maximum likelihood which are
character based analysis methods that treat every single site of the multiple alignment
independently. The second analysis used neighbor-joining/UPGMA method, a
distance method which summarizes the differences between sequences by calculating
a pairwise distance measure between all aligned sequences. The validity of each tree
obtained was checked by Bootstrapping (maximum likelihood analysis) and by
heuristic analysis of the UPGMA tree. Both trees were well supported by these
validity tests.
The phylogenetic tree in Figure 15 clearly delineates two major branch points
that correspond to metazoans and plants. The monocot plant genes form one
monophyletic group as do the mammalian genes. Another group that formed a
monophyletic dade was the insects.
The ZmTlk genes are ancient orthologs
The two classes of Tik genes in maize (ZmTlkl and ZmTlk2/3) are the result of the
allotetraploidization event that produced maize. Maize has three TLK genes unlike
Arabidopsis
and rice in which TLKs are single copy genes. The three maize genes
probably originate from duplication since they share high sequence homology, with
ZmTlk2 and ZmTlk3 being almost identical (96%) to each other and closely identical
(9 1%) to ZmTlkl in the coding regions. TOUSLED-like sequences had been mapped
to maize chromosomes 1L (position 1.12), 4c-L (position 4.05) and 5S (position 5.02)
by restriction fragment length polymorphisms (RFLP) using a eDNA probe from the
C-terminal of ZmTlkl that recognizes all three ZmTlk genes (Helentjaris, 1995,
Pioneer composite map). Maize chromosome arms 1L and 5S are duplicated
chromosome segments that resulted from the polyploidization event that yielded maize
and therefore gene syntenies that map to these regions are originally from the
individual maize progenitors. To determine whether the presence of two genes in the
ZmTlk2 and ZmTlk3 class of maize TLK genes is a result of a recent or ancient
duplication in the maize genome as put forward in the Introduction and Literature
Review section of this work (Figure 5), the maize Tlk genes were mapped to specific
loci in the maize genome. In the maize inbred line B73, the ZmTlk genes can be
differentiated based on restriction fragment length polymorphism of Tlk C- Tlk Zr
PCR products. The above PCR approach was applied to the oat-maize (oma) hybrid
lines. Each oma line contains the full oat chromosome complement and one maize
chromosome in addition.
ZmTlkl is present in the OMA line 1 (Figure 16) and is therefore indicative of its
presence on maize chromosome 1. Therefore, ZmTlkl is the maize TLK that maps to
1L position 1.12, with ZmTlk2 and 3 on chromosomes 4c-L and 5S. The
chromosomal position of ZmTlkl was confirmed by other PCR reactions (primer set
TLK A TLK B followed by Sad restriction digest) that supported the initial results.
UJA
A
S1c1
LIIL_C
TLZr
_ _ - U I
ZmTIkI
TJ..k_B
1JJ,.0
TJJL4
I
ZmTIk2
Ec?rI
TIkZr
I
LILB
Z m T 1k 3
TJjc
II]
'U
,\ff
omal
oma5 St-I
Ala
S60
5a
X4a
B73
no T omal
oma4
I.
uncut oat
{
ZmTlkI.*Ø*
uncut
.
cut oat{
ZmTIk2/3
cut
*
L
0
0
$
$
Figure 16 Mapping the ZmTlk chromosomal loci by PCR analysis of the oat-maize
hybrid lines.
(A) Genomic catalytic region of the ZmTlk genes. Primers and unique restriction
enzyme sites are shown. The unsequenced region of ZmTlk3 is indicated by a
fragmented box. (B) ZmTlkl is on maize chromosome 1. ZmTlk2 and ZmTlk3 cannot
be differentiated between at the C-terminal in S60. C- Zr PCR amplified products
from oat-maize addition (oma) lines 1, 4 and 5, maize inbred lines B73 and S60 (S60
is the maize parent used in generation of addition lines), St-i, the oat parent used in
generating the addition lines. Lanes are alternating PCR products followed by EcoRi
digested products of the same.
87
Specific loci could not be assigned for ZmTlk2 and 3 because the maize parent
used to generate the oat-maize hybrid panel was different from the B73 line used for
the libraries from which the ZmTlk gene clones were isolated. In S60, ZmTlk2 and 3
cannot be differentiated at the C-terminal (Figure 16). Additionally, PCR reactions
performed with primers other than the Tik C - TIk Zr pair (Table 3) yielded only oat
PCR products in oma4 and oma5. Sequence analysis of the Tlk C- Tlk Zr products of
ZmTlk 2 and 3 in S60 showed that they were almost 99% identical in that region as
compared to 96% in B73. It was interesting to note that the probe MTK4-2, which
contains a portion of intron 12 specific to ZmTlk2 in B73 and W22, recognizes two
distinct bands on an S60 maize genomic Southern.
Maize chromosome IL and SS are syntenic to each other. Based on the
sequence similarity between ZmTlk2 and 3 and the fact that ZmTlkl is on maize
chromosome 1L, it appears that ZmTlk3 is a recent duplication of ZmTlk2 and is
expected to be on chromosome 4c-L.
TLK genes in other grasses
Maize (Zea
mays mays L.)
is a member of the grass/Poaceae family and is
classified as a member of the PACCAD dade in which it is in the Panicoideae
subfamily, tribe Andropogoneae and genus Zea. Rice is also a member of the
grass/Poaceae family and is classified as a member of the Ehrhartoideae subfamily in
which is in the tribe Oryzoideae and genus Oryza. Rice is a distant relative of maize.
Other members of the Zea genus being also derived from the allotetraploidization
event that yielded maize, should have two classes of TLK genes with similar numbers,
like maize does if indeed the two ZmTLK classes are of ancient origin. To determine
whether TLK genes in other members of the grass family are more similar to maize or
rice in terms of number and type of genes, I examined the numbers of TLK genes in
other members of the genus Zea (Zea mays subssp huehuetenangensis, Zea mays
subssp mexicana, Zea mays subssp parviglumis, Zea diploperennis, Zea luxurians, Zea
perennis), the Andropogoneae tribe (all Zea species listed, Sorghum bicolor, and
Tripsacum dactyloides), and Poaceae family (Pooideae subfamily members wheat and
oat, Oryzoideae member rice, and the Panicoideae members Zea, Sorghum bicolor,
and Tripsacum dactyloides). Figure 4 is a phylogram that shows the relationship
between these grasses (taken from Kellogg, 1998).
TOUSLED-like kinase sequences were PCR amplified from the Zea species
using maize TLK primers (Figure 17 A). In Zea mays ssp. mays, there is a Sac 1 site
present in exon 13 of ZmTlkl but not in ZmTlk2 (and ZmTlk3 by implication) and as
such is used as a polymorphism to distinguish ZmTlkl -like genes from ZmTlk2/3-like
genes (map shown in Figure 16). Diagnostic restricition enzyme digests of the PCR
products ampilifed from the Zea grasses showed that there are two classes of TLK
genes in other Zea grasses as it is in Zea mays ssp. mays (Figure 17 B). The resulting
fragments from the enzymatic digest in both classes of TLK genes i.e. those that had a
Sad site and those that did not the Sad site, were subjected to direct sequencing (the
top two bands shown in Figure 17 B). Examination of the sequence electropherogram
data showed that some PCR fragments contained more than than one TLK fragments
since the electropherogram showed overlapping peaks in some regions indicating
heterogeneity in the fragment that was sequenced (appendix). The heterogeneity
could be as a result of duplicate genes or different alleles of the same gene being
amplified by the PCR reaction. However, for the Zea mays ssp. mays S60 line, the
heterogeneity level corresponded to that previously seen when PCR fragments of TLK
exons 15 and 16 and the intervening intron from maize chromosomes 4 and 5 were
sequenced. In addition, two separate lines for both Zea diploperennis and Zea mays
ssp. parviglumis were sequenced and in both cases, the same PCR fragments showed
heterogeneity. Based on these results it can be concluded that the heterogeneity
observed in the sequences were due to the prescence of different genes amplified in
the PCR reaction, however allelic variation cannot be emphatically ruled out in all the
heterogeneity cases observed. From the sequencing data (table in Figure 17 and
appendix), it appears that members of the Zea genus generally have three TLK genes
with one in the ZmTlkl class and two in the ZmTlk2/3 class. The exceptions are: Zea
luxurians which has one gene in each class, Zea mays ssp. huehuetenangensis which
has one gene in the ZmTlk2/3 class and two in the ZmTlkl class and Zea mays ssp.
parviglumis, which appears to have four TLK genes with two in each ZmTlk class.
ri'
mTlk
B
*
-
Zm T1k2/3 uncut
...
ZmTlkI Sad
products
C
ZmTlk2/3-like: no Sac! site
Sequenced with Tik A
ZmW22
ZinS6O
Zi
Zd
Zmh
Zmin
Zmp
Td
2 genes
2 genes less diverged than W22
I gene
2 genes
1 gene
2 genes
2 genes
2 genes
ZmTIkl-like: Sac! site
Sequenced with Tik A
1 gene
1 gene
1 gene
1 gene
2 genes
I gene
2 genes
2 genes
Figure 17 PCR amplification of Tik genes (exons 12-14 and intervening introns) from
Zea species.
In the Zea genus, numbers and pattern of TLK genes approximate that in Zea mays
subsp. mays. (A) Zea genus PCR amplified products using primers Tlk A and Tik
XYr. (B) Sac 1 digests of PCR amplified products. Abbreviations used in figure are:
ZmW22 = Zea mays mays (maize) inbred W22, Zl = Zea luxurians, Zd = Zea
diploperennis, Zmh = Zea mays huehuetenangensis, Zmm = Zea mays mexicana, Zmp
= Zea mays parviglumis and Td Tripsacum dactyloides. nD refers to a no template
control. All grasses shown in this figure are from the Zea genus with the exception of
Tripsacum dactyloides which is a close relative of the Zea species.
(C) Table shows putative number of genes as determined by examining sequence
electropherograms (appendix) obtained from sequencing the PCR fragments (Sac 1 cut
and uncut) indicated by the * symbol.
91
ZntB7 3- ZmTl k2
Zrnh- class2
Zl-class2
Sorghum
Zi -clasal
ZmS6O ZmTlkl
Zd2 classi
ZmW2 2- ZmTlkl
Zd]. class).
Zmm- classl
Hordeum
ACATTCTGCACCG TCCTAGAATATTGCAGTGGTATTATATGTTTTTATGATCTTCGGAA
ACATTCTGCACCG -TCCTAGAATATTGCAGTGGTATTATATGTTTTTATGATCTTCAGAA
------------------------------------------- TTTTATGATCTTCAGAA
ACATTCTGCACCG TCCTAGAATATTGCAGTGGNCTTATATGTTTTTATGACCTTCGGAA
ACATTCTGCACTGGTCCTAGTATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTG-TCCTAGAP.TATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTG-TCCTAGAATATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTGGTCCTAGAATATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTG- TCCTAGAATATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTGGTCCTAGAATATTGCAGTGGTATTATATGTTTTTATGACCTTCTAAA
ACATTCTGCACTG TCCTAGAATATTGCAGCGGTATATAATAATTTCTTAA.ATTTCTTTG
***
ZmB73 ZmTlk2
Zmh-class2
Zi class2
Sorghum
Z 1-class).
ZmS6 0- ZmTlkl
Zd2 classl
ZmW2 2- ZmTlkl
Zdl class).
Zmm- class 1
Hordeum
ZmB7 3- ZmTlk2
Zmh ci as s2
Zi class2
Sorghum
Z 1- class 1
ZmS6 0- ZmTlkl
Zd2 -classl
ZmW2 2- ZmTlkl
Zdl -classi
Zmm- class 1
Hordeum
Figure 18
species.
*
*
***
TCCTGATTGATATGACAA ------ CATAACAAACTC- -TTTTATTTGTTCGCTTTTCCT
TCCTGATTGATATGACAA ------ CATAACAAACTC- - -TTTTATTTGTTCGCTTTTCCT
TCCTGATTGATATGACAA ------ CATAACAAACTC- - -TTTTATTTGTTCGCTTTTCCT
TCCCAATTGATATGACAA ------ CGTAACAG- -TA- -TTTTATTTGTTCGCAGTTCCT
TCCTGATTGATATGACAATGACAACATAGATAAGCCAGGTTTTATTTGTTCGCTGTTGCC
TCCTGATTGATATGACAATGACAACATAGATAAGTCAGGTTTTATTTGTTCGCTGTTGCC
TCCTGATTGATATGACAATGACAACATAGATAAGTCAGGTTTTATTTGTTCGCTGTTGCC
TCCTGATTGATATGACAATGACAACATAGATAAGTCAGGTTTTATTTGTTCGCTGTTGCC
TCCTGATTGATATGACAATGACAACATAGATAAGTCAGGTTTTATTTGTTCGCTGTTGCC
TCCTGATTGATATGACAATGACAACATAGATAAGTCAGGTTTTATTTGTTCGCTGTTGCC
ACATTTTGACTGTCTAAA ------ TTTGGTTCTGTCATTTTTGTTTTGCTTGGAGT- -CT
*
*
* *
**
*
***
**** * *
*
*
GATACCTGTATGCGGATGACTTCATTCTGTAGTCATGCTAACATAAATTC -ATAATTTGG
GATACCTGTATGCGGATGACTTCATTCTGTAGTCATGCTAACATAAATTC -ATAATTTGG
GATACCTGTATGCGGATGACTTCATTCTGTAGTCATGCTAACATAAATTC-ATAATTTGG
GATATCTGTCTGCGGAGGACTTCATTATGTAGTCATGCTAACATAAACTC -ATAACTCAG
GATACCTATCC CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC -ATAATTCAG
GATACCTATCC- CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC-GTAATTCAG
GATACCTATCC-CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC-GTAATTCAG
GATACCTATCC CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC -GTAATTCAG
GNTACCTATCC-CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC-GTAATTCAG
GATACCTATCC CAGAGGATTTCATTCTGTAGCCATGCTAGCATAAATTC -GTAATTCAG
TATGCCATTCT- - -GATGA-TGCACATTGCC-TCATGTTTCCTTTAGTTTTGTAATACAG
*
*
*
** ** * **
**
**** * * * * *
***
*
Alignment of Tik intron 12 sequences from eleven different Poaceae
Poaceae species and genes analyzed by this alignment are: maize i.e. Zea mays subsp
mays inbred lines B73, S60 and W22 (ZmB73-ZmTlk2, ZmS6O- ZmTlkl and
ZmW22- ZmTlkl), Zea mays huehuetenangensis T1k2 (Zmh-class2), Zea
diploperennis two lines (1 and 2) sequenced, Tlkl (Zdl-classl and Zd2-classl), Zea
mays mexicana Tlkl (Zmh-classl), Sorghum bicolor (Sorghum) and Hordeum vulgare
(Hordeum). This sequence alignment indicates conservation of intron 12 in these
species. Initial characterization of Tik genes into class 1 and class 2 is based on
restriction enzyme polymorphisms. The single Sorghum bicolor Tik gene is more
similar to ZmTlk2 than to ZmTlkl. All sequences obtained for Poaceae species
during this study are included in the appendix.
ZmB73 ZmTlk2
Zmh class2
Zl claas2
Sorghum
Zl classl
ZmS6O ZmTlkl
Zd2 -classl
ZmW2 2- ZmTlkl
Zdl -classl
Zmm- classl
Hordeum
AAG-TGTTAGA- -GATACTGTCTTACCGAACTTTAGGCAT- -ATATTGTTATTGGCCACA
AAG TGTTAGA- -GATACTGTCTTACCGAACTTTAGGCAT -ATATTGTTATTGGCCACA
AAG-TGTTAGA- -GATACTGTCTTACCGAACTTTAGGCAT- -ATATTGTTATTGGCCACA
AAG TGTTAGATAGATACTGTCTTAC GAACTTTAGGCAT - -ATATTGTTATTGGCCACA
AAG-TGTTAGATAGATATCATATCACTGACCTTTGGGTAT- -CTATTGTTATTAGCCACA
AAG-TGTTAGATAGATATCATATCACTGACATTTGGGTAT- -CTATTGTTATTAGCCACA
AAG- TGTTAGATAGATATCATATCACTGACATTTGGGTAT- CTATTGTTATTAGCCACA
AAG-TGTTAGATAGATATCATATCACTGACATTTGGGTAT- CTATTGTTATTAGCCACA
AAG-TGTTAGAT- - -ATCATATCACTGACATTTGGGTAT- -CTATTGTTATTAGCCACA
AAG- TGTTAGATAGATATCATATCACTGACCTTTGGGTAT- CTATTGTTATTAGCCACA
APAATGTTAGTTAGATTTTGCGCCAGGCATGGTGTAGCCTTGATACATCTATCTATGATG
******
**
ZmB73 ZmTlk2
Zmh- class2
Zl -class2
Sorghum
Zl -classl
ZmS6O -ZmTlkl
Zd2 -classl
ZmW22 - ZmTlk].
Zdl -classl
Zmm-classl
Hordeum
Zmh - c las s2
Zl
class2
Sorghum
Zl class 1
ZmS 60- ZmTlkl
Zd2 clas sl
ZmW2 2- ZmTlkl
Zdl class 1
Zmm- class 1
Hordeum
*
*
*
***
**
*
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCCAGTCAAAGAACACAG
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCCAGTCAAAGAACACAG
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCCAGTCAAAGAACACAG
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCCAGTCAAAGAACACAG
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCCAGTCAAP.GAACACAG
TACATTCATGTTTA ------------ GAAATTGTTCTATTTTGCAAGTCAAAGAACACAG
AACTCTTAC -TTCATTAAACTTATTCCAGTTTTTACTATTTAGCCA-T -AGTCGCCAGTA
**
*
*
*
**
****
*
*
*
*
*
GTGGTTCTTAGAT-AA- - -AGTAATAGCTGCTGACCCATATAAAAATGAAGTGAATGCA
GTGGTTCTTAGAT -AG ----- GTAATAGCTGCTGACCCATATAAAAATGAAGTGAATGCA
GTGGTTCTTAGAT -AG ----- GTAATAGCTGCTGACCCATATAAAAATGAAGTGAATGCA
GTGGTTCTTGGAG - AG ----- GCAATAGTTGCTGACCTATATAAAAATGAAGTGAATGAA
GTGGTTCTTGGAG - AGGTAATACAATAGTTGCTGATTCTTATAAAATTGAAATGAATGCA
GTGGTTTTTGGAG - AGGTGATACAATAGTTGCTGATTCATATAAAATTGAAATGAATGCA
GTGGTTCTTGGAG - AGGTAATACAATAGTTGCTGATTCATATAAAATTGAAATGAATGCA
GTGGTTCTTGGAG AGGTAATACAATAGTTGCTGATTCATATAAAATTGAAATGAATGCA
GTGGTTCTTGGAG -AGGTAATACAATAGTTGCTGATTCATATAAAATTGAAATGAATGCA
GTGGTTCTTGGAG -AGGTAATACAATAGTTGCTGATTCATATAAAATTGAAATGAATGCA
G GTGTCCTGGAGTAAACAGAGATATAAATGTTTAAATTTG GGAACTAAAAGGGACATC
*
ZmB73 ZmTlk2
*
TGCGTTTGTGTTTATAGTTCTTGGATGAATTTGTTTCATTTTGCCAATAAAAAAACTGTG
TGCGTTCATGTTTATAGTTCTTGGATGAATTTGTTTCATTTTACCAATAAAAAAACTGTG
TGCGTTCATGTTTATAGTTCTTGGATGAATTTGTTTCATTTTACCAATAAAAAAACTGTG
TGCATTCATGTTTATAGTTCTTGGATGAATTTGTTCCATTTTGCCAGTCAAAAA- TTGAG
*
ZmB73 -ZmTlk2
*
*
*
*
**
***
*
**
*
*
*
**
*
**
*
*
Zl
TAAACTGATGCTA-AATAGATTTGGTGATGGTTTATACAATTATTTCTCCCACATTTACC
TAAACTGATGCTA-AATAGATTTGGTAATGGTTTATACAATTATTTCTCCCACATTTACC
TAAACTGATGCTA-AATAGATTTGGNAATGGTTTATACAATTATTTCTCCCACATTTACC
TAAACTGACGCTT CATAGATTTGGTAATGGTTTATACAATTATTTCTCCCACATTTGCC
ZmW22 -ZmTlkl
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
TAAACTGATGCTT-CATAGATTTGGTAATGGTTTA ----------------------- CC
Zmh clas s2
class2
Sorghum
Zl-classl
ZmS6O-ZmTlkl
Zd2-classl
Zdl-classl
Zmm-classl
Hordeum
TAAACC-ATGCTTCCAGTGATTTGGTCATGGATTCTTACAGTTACAGTC --------- CA
*****
*
***
*
*******
**** **
*
Figure 18 Alignment of Tlk intron 12 sequences from eleven different Poaceae
species (continued).
93
ZmB 73- ZmTlk2
Zrnh c las s2
Zl ci as s2
Sorghum
Zi classi
ZmS6O ZmTlkl
Zd2 -classi
ZmW22 ZmTlkl
Zdl classi
Zmm-classl
Hordeum
ATTGGTCAGAATAGGATAGGTTGTCAATGGGGGAAAAAAGGCAGGGGCAACATGCTGTT
AATTGGTCAGAATAGGATAGGTTGTCAATGGGGAAAAAAAGGCAGGGGCAACATGCTGTT
AATTGGTCAGAATAGGATAGGTTGTCAATGGGGAAAAAA- GGCAGGGGCAACATGCTGTT
CATTGGTCAGACTAGGATAGGTTGCCAATGGGGAAAAAA GGCAGGGGCAACATGCTGTT
CATTGGTCAGACTAGGATAGGTTGTCAP.TGGGGGAAAAA-GTCAGGG _____________
CATTGGTCAGACTAGGATAGGTTGTCAATGGGGGAP.AAA-GTCAGGG _____________
CATTGGTCAGACTAGGATAGGTTGTCAPTGGGGGAAAAA-GTCAGGG _____________
CATTGGTCAGACTAGGATAGGTTGTCAPTGGGGGAAAAAAGTCAGGG _____________
CATTGGTCAGACTAGGATAGGTTGTCATGGGGGAAAA-GTCGGG _____________
CATTGGTCAGACTAGGATAGGTTGTCA)TGGGGGAAAAA _____________________
CATT-GTCA-TTTATAATC- -TTGTAAAATATGGATCA- -CCCATACCPiCATTATGAG
*** ****
ZmB73 -ZmTlk2
Zmh-class2
Zi class2
Sorghum
Zi classi
ZmS6O ZmTlkl
Zd2 -ciassi
ZmW22 - ZmTlkl
Zdl-classl
Zmm-classl
Hordeum
**
**
***
**
*
*
*
GACCGGATTTG ----- TATCCCATCCTCTGTCTCGACAAT- -A1CAAGCAAGTTGACAAA
GACCGGATTTG ----- TATCCCATCCTCTGTCTCGACAAT- -AACAAGCAAGTTGACAAA
GACCGGATTTG ----- TATCCCATCCTCTGTCTCGACAAT- -AACAAGCAAGTTGACAAA
TTTG
------- TTTG
TTTG
------- TTTG
TTTG
----- TATCCTATCCTCTGTCTCAACAAT----- TATCCTATCCTCTGCCTCAACAAT----- TATCCTATCCTCTGTCTCAACAAT----- TATCCTATCCTTTGTCTCAACAAT----- TATCCTATCCTCTGTCTCAACA1T-
-ACCAGACAAATTGACAAA
-ACCAGACAAATTGACAAA
-ACCAGACAAATTGACAAA
-ACCAGACAAATTGACAAA
-ACCAGACAAATTGACAAA
CTTAAGATGTACGGAAAATAACTGCC -AThTTTGATCACT -ACATAAGAAGCATACATA
Figure 18 Alignment of Tik intron 12 sequences from eleven different Poaceae
species (continued).
94
TOUSLED-like kinase sequences were PCR amplified from non-Zea grasses
using maize TLK primers (Figure 17 and Figure 19). Diagnostic restricition enzyme
digests of the PCR products showed that there are two classes of TLK genes in
7'ripsacum dactyloides (Figure 17), Avena sativa and Triticum aestivum (Figure 19) as
is the case in the Zea grasses. However, there is one class of TLK genes in Sorghum
bicolor and Hordeum vulgare. The resulting fragments from the enzymatic digest of
both classes of ILK genes in Tripsacum dactyloides and of the single TLK classes of
Sorghum bicolor and Hordeum vulgare were subjected to direct sequencing.
Examination of the sequence electropherogram data showed that both classes of TLK
genes in Tripsacum dactyloides contained more than than one TLK fragments since
the electropherogram showed overlapping peaks in some regions indicating
heterogeneity in the fragments that were sequenced (appendix). These results suggest
that Tripsacum dactyloides has four TLK genes with two in each ZmTlk class.
Examination of the PCR and restriction enzyme digest products (Figure 19) and
sequence results of the PCR fragments (appendix) indicate that Sorghum bicolor and
Hordeum vulgare each have one TLK gene. The sorghum gene is more similar to the
ZmTlk2 gene than to the ZmTlkl gene based on intron 12 sequence analyses (Figure
18).
From examination of the PCR products and restriction enzyme digest products
of the PCR, it appears that both Avena sativa (oat) and Triticum aestivum (wheat) have
at least three or more genes (Figure 19). Triticum aestivum appears to have at least
one TLK gene in each class (Figure 19).
95
Sb
Ta
Hv
As
ZmW22
LJ
4
ZmTlk2/3 uncut
ZmTIkl Sad products
Figure 19 PCR amplification of Tik genes from non-Zea Poaceae families.
Non- Zea grasses have varied number of Tik genes in their genomes.
(A) Tik A Tik XYr PCR amplified products from a selection of grass families.
(B) Sac 1 digested PCR amplified products. Abbreviations used in figure are: Sb =
Sorghum bicolor, Ta = Triticum aestivum, (bread wheat) Chinese Spring variety, Hv =
Hordeum vulgare, (barley) opata variety, As = Avena sativa (oat) Starter I variety and
ZmW22 = Zea mays mays (maize) inbred W22.
Analysis of the mRNA expression levels of the TLK genes in
various maize tissues
ZmTlk expression is higher in dividing tissues
The maize ZmTlk genes are expressed in all tissues examined but there are
tissue and temporal differences in their expression.
Arabidopsis TSL
mRNA has been
shown to be constitutively expressed in all tissues of the plant with the highest level of
expression occurring in the developing floral buds (Roe et al., 1993). To determine
the TLK expression levels in maize tissues and whether the expression pattern of the
ZmTlk mRNA parallels that of Arabidopsis, a semi-quantitative RT-PCR approach
was used to assay TLK expression in various maize tissues (modified from Nebenfuhr
and Lomax, 1998).
The maize TLKs are detected in all tissues examined (Figure 20) but expression
levels differ substantially among the various organs. The semi-quantitative RT-PCR
protocol and analysis method used to determine levels of expression in various tissues
is described in the Materials and Methods chapter of this work. Figure 21 shows an
example of the semi-quantitative RT-PCR results. Serial dilutions of the cDNA
prepared from each tissue were used as template for the PCR amplification. As
substrate level becomes limiting, the amplified product's concentration becomes
proportional to the template concentration. Primers specific for maize Actin 1 cDNA
were added to each PCR as an internal control of RNA quality and level and for
normalization purposes.
97
Sac I
1J,A
ZmTlk 1
Regulatory region
Catalytic region
!r
ZmTIk 2
Regulatory region
Catalytic region
/
,0
ZmTIkl
\
.c
çr
I
ZmTlk2/3 -+
c\
.
=
= =
Figure 20 Tissue distribution of ZmTlk transcripts as analyzed by RT-PCR and
restriction enzyme digestion.
(A) The cDNAs of ZmTlkl and ZmTlk2 are shown as schematic figures. Primers
used and the Sac 1 site that differentiates between them is indicated. (B) shows that
transcripts of both maize Tlk classes are present in all tissues examined.
20 DAP endosperm
4X
MX 256X
16X
12 DAP endosperni
17 DAP eiidosperm
_-
4X
MX 256X 4X
16X
ZmTlk2/3 +
16X MX 256X
Aclin +
ZmTIkl
I
Actin
*
Normalized
ZmTlk2/3
20-DAP
cDNA
dilutions
1/4= 0.25
1/16
1/64
1/256
ZmTlk2/3
22309
17950
7159
2114
Actin
bandi
1536
1911
1105
579
C
ZmTlkl
3088
3233
1581
531
Actin
bands2
3389
4442
4604
3067
transcript
abundance
ZmTlk2/3/cActin
4.529746193
2.825436802
1.253984936
0.579813494
Combined
Actin
20-DAP
Normalized
4925
6353
5709
3646
ZmTlkl
transcript
abundance
ZmTlkl/cActin
0.627005076
0.508893436
0.276931161
0.145639057
ZmTlk2/3 transcript amount
y = 36.954x + 0.5426
R2 = 0.9886
3
tlk2/3
1.5
1
.--tIkl
Linear (t1k213)
Linear (tiki)
0.5
y=5.8421x+0.1507
-
0
0.07
0.06
0.05
0.04
0.03
feD NA] dilutions
0.02
0.01
R2 =
0.9697
0
ZmTlkl transcript amount,
Figure 21 Calculating the mRNA transcript levels of ZmTLK genes in maize tissues.
(A) RT-PCR reactions using four-fold serial dilutions of templates from maize W22
12, 17 and 20 days after pollination (DAP) endosperm. (B) The normalization process
for the 20-DAP serial dilution quantitation values. (C) shows how the data was
extrapolated to obtain the transcript levels. The normalized 20-DAP values are
plotted. From the equation of the curves, the value of y, when x =1 is the relative DNA
fluorescence value used in plotting the graphs in Figure 22 through Figure 24.
Figure 22 shows that the ZmTlk transcripts accumulate to a higher level in
immature tissues in which cell division is occurring at a rapid rate as compared to
mature tissues in which cell division is low or has halted substantially. The ZmTlk
expression levels were also compared to the expression levels of a maize cyclin B 1
gene, CycZmel. CycZmel expression correlates with tissues in which cell division is
occurring and is virtually absent in non-dividing cells. Figure 22 shows that while
CycZmel is indeed more highly expressed in rapidly dividing tissues and almost
absent from non-rapidly dividing tissues, ZmTlks expression is higher than CycZmel
in both types of tissues. However, ZmTlks are definitely more highly expressed in
dividing tissues.
A comparison of ZmTlk expression in vegetative and floral tissues (Figure 22
and Figure 23) shows that ZmTlks are more highly expressed in the floral buds than in
the vegetative tissues. In vegetative tissue, Tlk expression is higher in roots than in
leaves.
100
12x
10
8x
0
6x
0
I)
4x
z
2x
Ox
6
4
1e/
Ii)ividing nize Tissues
Non-Dividing nnize Tissues
Figure 22 The mRNA expression level of ZmTlkl and ZmTlk2/3 is higher in
dividing than in non-dividing maize tissues.
wp refers to week-old plant. 6wp ear refers to an ear from a six week-old plant.
Cyclin Bi (CycZmel) is a mitotic cyclin and is used as a dividing tissue marker in
these experiments.
101
ZmTlk expression correlates with S-phase and is highest in
endoreduplicating tissues
The high expression of Zmllk genes in dividing tissues suggests a role for
ZmTLKs in mitosis or DNA synthesis. To test this hypothesis, I compared ZmTlk
expression levels with the transcript levels of a mitotic cyclin, CycZme 1 and an Sphase gene, histone H3 in tissues that were dividing or undergoing endoreduplication.
The maize leaf and root can be spatially divided into dividing and expanding
regions. Figure 23 compares the difference in expression between the dividing regions
of the root tip and leaf and the expanding region of the root and leaf and shows that
Tik transcripts are higher in dividing than in non-dividing tissue. This correlated with
the pattern of expression of CycZme 1, a maize cyclin B 1 gene (Figure 23).
Generally the ZmTlk2i3 class shows slightly higher expression than the ZmTlkl
class in all tissues examined, however, in the root dividing tissue, ZmTlk2/3
expression is 2X that ofZmTlkl and in the root expanding tissue ZmTlk2I3
expression is 4X that of ZmTlkl. ZmTlk2/3 expression in expanding tissues of the
leaf and root is 0.5X the expression level in dividing tissues of the leaf and root.
Though the expression pattern of the ZmTlk genes paralleled that of CycZmel
in the leaf and root tip tissues examined, however subsequent analysis showed that the
niRNA levels of the maize Tik genes parallel that of maize histone H3, an S-phase
gene, more closely than that of CycZmel, an M-phase cyclin (Figure 24).
102
A
1.8
1.6
1.4
1.2
(8
0.8
0.6
0.4
0.2
leaf, dividing
region
leaf, expanding
region
root, dividing
region
root, expanding
region
Figure 23 Difference in the expression levels of maize Tik and cyclin Bi genes in
dividing and expanding root and leaf segments.
(A) region of maize seedling root tip to 2 mM away, actively dividing. (B)area of a
maize seedling root 10 mM away from the tip undergoing expansion. Both images
are taken at 20X magnification. (C) Graph shows that while the cyclin Bi is on in the
dividing section but not the expanding region, the tik genes are being expressed in
both regions with higher expression evident in the dividing region.
103
35
30
Co
25
J
i-15
0)
w10
ZmTLKI
Figure 24
ZmTLK2/3
Cyclin BI
Histone H3
Patterns of ZmTlk expression in developing endosperms.
Expression levels of the ZmTlk2/3 class increase as the endoreduplicating cycle
progresses and is most highly expressed in 20-DAP endosperm tissue. In maize
endosperm tissues, mRNA accumulation of TLK, Cyclin B 1 and Histone H3 are
similar during the early stages of development (9-DAP). As the tissue matures, the
expression pattern observed for each gene changes with progression of the
endoreduplication cycle. mRNA transcript accumulation of the maize Tik genes more
closely parallel that of Histone H3, an S-phase gene than a maize Cyclin Bi,
Cyczmel, an M-phase gene. N refers to endosperm tissue and E refers to embryo.
104
Generally, the ZmTlk2/3 class transcript levels are higher than the ZmTlkl
transcript levels. The most striking differences in expression between the two classes
were the observed up-regulation of ZmTlk2/3 in the developing endosperm, especially
in the endosperm tissues 20 days after pollination (Figure 24). ZmTlk2/3 is 6X higher
than ZmTIkl in those tissues as compared to being 1 .5X higher in other tissues. In
addition, in the root expanding tissue, ZmTlk2f3 expression is 4X that of ZmTlkl
(Figure 23).
Investigation of ZmTIk Function
Characterization of TUSC-TLK lines
To investigate the function of the ZmTlk genes in maize, we obtained 12 maize
families potentially harboring Mutator insertions in TLK genes (trait utility system for
corn, i.e. TUSC lines) from Pioneer Hi-Bred and analyzed them. The TUSC families
were planted at the Botany and Plant Pathology field laboratory and leaf tissues were
collected from each plant for genotyping. TUSC lines were genotyped by analysis of
their PCR products for Mu insertions into TLK genes. PCR analysis was followed by
Southern blotting of the PCR products and probing the blot with radiolabeled TLK
probes to investigate the possibility of Mutator insertions in these lines. Figure 25
shows that PCR products visible on ethiduim bromide stained gels were not the same
size as the bands which hybridized with the radiolabeled probes on Southern blot of
the same gel. Based upon Southern blotting, five putative maize TLK mutant families
were identified: TUSCTII121, TUSCTJk129, TUSCTIk13O, TUSCTIk135 and
105
TUSCT1k145 but the Mu insertions could not be verified. Therefore, no maize
mutants for the ZmTlk genes were obtained.
106
Regulatory Region
\y'i
V
W
X
XY
4-
Vfsapl
A
Xrl
Y
z
4B
4-
4-
Zr
T1k4
3
1
Family
Mu B PCR
products/bp
Mu B PCR probed
cEAR
/bp
Positives/family
tlk-121
300,700
580
7/11
0/12
tlk-126
tlk-129
300,380
tlk-130
9/11
250, 380
250
974
tlk-135
224
750
tlk-145
230,325,400
350,450
2/9
5/12
Figure 25
3/8
The putative maize ilk mutant families.
(A) The maize TLK 2 protein domains and the corresponding genomic DNA region of
the catalytic region with primers used for ilk mutant identification are shown.
(B) Mu - Tlk B PCR amplification products sizes are different from the fragment sizes
obtained when the same PCR products are blotted unto a nylon membrane and probed
with a radiolabeled portion of the maize TLK catalytic region.
107
ZmTLK2 is a functional homolog of Arabidopsis TSL
The complementation of tsl mutants with wild-type genomic TSL gene via
Agrobacterium tumefaciens-mediated transformation of the mutant plants (Roe et al.,
1993) provided evidence that the TOUSLED (is!) phenotype occurred as a result of
mutation of the Arabidopsis TSL gene. The tsl mutation has been described in detail
in the Introduction and Literature review chapter and listed in Table 6 are the
Arabidopsis floral and vegetative morphology affected by the tsl-1 mutation. The
predicted proteins of ZmTLK1 and ZmTLK2 are 83% identical to AtTSL at the
catalytic region and 45% and 49% identical respectively to AtTSL at the N-terminal
regulatory domain. Such sequence conservation may represent conservation of
function of these TLK proteins in maize and Arabidopsis even though the two plants
have divergent morphology. The N-terminal being more structurally conserved than
sequence conserved may however confer different functions via different interactions
partners to these kinases, however the kinase region of the ZmTLK proteins should be
functionally equivalent to AtTSL.
To investigate the functional significance of the ZmTlk genes, the tsl-1 mutant
of Arabidopsis
(ecotype Wassilewskija, Ws) was transformed by a sense construct of
the ZmTlk2 cDNA (pFGCTLK) and also by a chimeric sense construct of the fused
TSL N-terminal and ZmTIk2 C-terminal (pFGCnTSL-cTLK). The chimeric construct
was necessary in the event that the N-terminal of ZmTlk2 was unable to interact with
the AtTSL partners at the N-terminal and therefore could not complement the tsl-1
mutation. The cDNA for each construct was introduced into the binary vector
pFGCS941 in such a way that expression of ZmTlk2 was driven by the constitutive
cauliflower mosaic virus (CaMV) 35S promoter.
Table 6
plants.
Phenotype of the tsl-1 mutants compared to wild-type
Arabidopsis thaliana
Wild type
Arabidopsis Plants
Phenotype of (si-i mutants
Number of Rosette leaves before
plant flowers is 5.9
In general tsl-1 plants are delayed in
flowering by about 1 week as compared to
wild type plants.
Number of leaves produced in vegetative
rosette of tsl-i (7.4) is l.5X that of wild
type siblings before flowering
Wild type leaves are less serrated
Rosette leaves more serrated
Secondary axes number about 2.4
Cauline leaves unfold away from
the axils
Four sepals
More secondary axes from axils of
cauline leaves, 4.2 in tsl-1.
Cauline leaves curl up tightly around new
axils
Fewer sepals (2-4 instead of 4)
Four petals
Fewer petals (0-4 instead of 4)
Six stamens
Fewer stamens (0-5 instead of 6)
Organs are regularly shaped and
well defined
Organs are often misshapen and reduced
in size or filamentous
Gynoecium is fused (bicarpellate)
Stigmatic tissue is always present
Gynoecium always split and stigmatic
tissue is seldom present.
tsl-1 female sterile at 22°C
109
Arabidopsis tsl-1 and wild-type Ws Arabidopsis plants carrying the resulting
plasmids were generated by Agrobacterium tumefaciens-mediated transformation via
the floral dip method (Clough and Bent, 1998) and selected by Basta resistance (Lutz
et al., 2001). All transgenic lines obtained were tested for the insertion of the
transgene in their genome by PCR, using primers specific for each construct in
addition to being selected for Basta resistance. Using PCR, the genetic background of
the transgenic lines was determined. For the tsl-1 allele, primer pair RB and RE and
for the wild-type TSL allele, primer pair Vfsapl and RE were employed in the PCR
analysis. An example of the genotype results is shown in Figure 27. Using RNA
prepared from rosette leaves, RT-PCR was performed to confirm that transgenic plants
carrying the constructs expressed the transgene (Figure 28).
Table 7
Arabidopsis Transgenic Lines used in this study.
Transgene
Original
transformed
line
N-terminal
C-terminal of
of gene
gene
transformed transformed
PFGC5941
tsl-1 in
Wassilewskija
35::TSL
(pFGCTSL)
tsl-1 in
TSL
Wassilewskija
TSL
35S::TLK2
(pFGCTLK)
tsl-1 in
ZmTLK2
Wassilewskija
ZmTLK2
35S::nTSL-cTLK
(pFGCnTSLcTLK)
tsl-1 in
TSL
Wassilewskija
ZmTLK2
-
Empty binary
vector
control
Full-length
TSL
CDNA
control
Full-length
ZmTLK2
cDNA
Chimeric
construct of
TSL and
TLK
110
Saot(6)....
Sail (563)
Pstl
HInoll (727)
Hinoll (754)
-Cl.l(830)
HInell (563:
ooJ(563)
EooRl (1224)
MAS3
BAR
_Bg III (1610)
ptlAS2
Hinoll (2135)
P350
-Bglll (2356)
Xhol (2562)
Aaoi (2573)
.?ool (2643)
Noti
0
pFGC5941
go
(11407 bp)
1
Cl (2649)
I 'çji (2659)
I Swat (2666)
mdlii (3172)
CHSR
Sp.i (3635)
SpOt (3912)
sit (3919)
an*ii (4030)
Avrii (4034)
Noti (7556)
Xbal (4044)
Pool (4053)
Xnal (4063)
Hinoli (7113)
Spot (4069)
Apal (4697)
PotI (4797)
5pM (4803)
Hmndlli (4809)
Notl(6024)
Sphl(5142)
Hinoll(5711)
Swal
B
pFGCTSL
I
Sapi
I
NdeI
NdeI
Xba I
pFGCTLK
SgrA I
Swa 1/BsaA I
Xba I
pFGCnTSL-cTLK
NdeI
Figure 26
SapI
STAI
Transgenic constructs used for transforming Arabidopsis plants.
(A) Binary vector pFGC5941. P35S represents the CaMV 35S promoter. Map and all
information for this vector were obtained from the plant chromatin database
(www.chronidb.org/plasrnids/new vector).
(B) Schematic representation of the Arabidopsis TSL and maize ZmTLK constructs
used in the complementation of the tsl-1 mutation. The empty binary vector was used
as the negative control in these experiments.
111
A
ArabidopsisTSLgenomic
I I I I I I I I I iI I I I I
RB
-,
TI RE
Arabidopistsigenomic
SwaIIBsaAl
pFGCnTSL-cTLK cDNA
I
111111 III I I I I II I
Tf
4
Arabidopsis
SajI
Nde I
B
Transgenic
Line
16-2
16-4
16-6
16-9
16-16
16-23
16-24
16-25
16-27
16-28
16-30
16-39
16-46
16-49
16-50
16-53
16-54
1655
Figure 27
tsl-1
allele
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
XbaI
Wild-type
TSL
allele
-
Transgenic
nTSL-TLK
SgMl
Phenotype
allele
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
Partial rescue
is!-]
Partial rescue
Partial rescue
is!-]
is!-]
Partial rescue
is!-]
An example of genotyping of transgenic lines by PCR analysis.
(A) schematic representation of the Arabidopsis TSL wild-type and tsl-1 mutant
alleles. The triangle in the tsl-1 schematic represents the T-DNA insertion in TSL
wild-type responsible for the tsl-1 mutation. Primers used are indicated. (B) results
are shown for T2 plants of pFGCnTSL-cTLK 16. The + symbol denotes the presence
of the tested gene. The - symbol indicates absence of the tested gene.
112
Sw I/BsaAl
A
Tjf
Xbal
v_fI
pFGCTSL
11
I
Ndel
SwaI/BsaAI
Tf
Sa1
NLI
Xbal
Vf1
pFGCnTSL-cTLK
I
NdeI
Sai1
si
Xbal
pFGCTLK
SgrAI
B
Tsl 15fRE
Detected mRNA
AtTSL
TslI5fXr4
nTSL-cTLK
PCR primers
Tik A Zr
ZmTLK2 and nTSL-cTLK
Figure 28 Expression of Transgenes.
RT-PCR was performed to confirm transgene expression in selected transgenic plants.
(A) Schematic diagram of transgenes. Primer positions are indicated. (B) RT-PCR
results indicated that the transgenes were expressed in plants that showed rescued tsl
phenotype. Ws cDNA: cDNA from an untransformed wild-type Arabidopsis
Wassilewskija plant. tsl-l: cDNA from an untransformed tsl-1 plant.
113
35S: :nTSL-cTLK Transformants
Eighteen Basta resistant transgenic lines corresponding to independent
transformation events (Ti) harboring the pFGCnTSL-cTLK sense construct were
obtained (Table 8). Of these 18 lines, 14 were TSL/TSL, three were heterozygous
TSL/tsl-1 and one was homozygous for the tsl-1 allele. The homozygous Ti plant
(pFGCnTSL-cTLK#16) showed a partially rescued tsl-1 phenotype. The plant was
able to set seed at 22°C and rosette leaves were phenotypically wild-type. However,
the number of organs in the flowers, not including the gynoecium, was less (9
11)
than in wild-type flowers (14) but slightly more than in tsl-1 plants (7-9). The
pFGCnTSL-cTLK#16 plant was allowed to self pollinate and the second generation
(T2) progeny was examined for phenotype and the presence of the maize transgene.
This line segregated for a single locus (3:1) of the transgene with 14 Basta-resistant
plants to four non-Basta-resistant plants.
The T2 plants of this line that had the transgene (e.g. pFGCnTSL-cTLK#16-2)
displayed the same phenotype of partial rescue of the tsl-1 mutation (Figure 29
through Figure 31 and Table 9 and Table 10) as the TI plant did. T2 plants that did
not have the transgene (e.g. pFGCnTSL-cTLK#16-55) showed a complete tsl-1
phenotype.
114
Table 8
Analysis of Transgenic Arabidopsis
Ti
Lines.
200 300 plants were transformed with each construct. These are TO plants. + refers
to the wild-type Arabidopsis TSL allele. Number in brackets in the genotype colunm
refers to the number of plants obtained for that genotype.
Line
Basta tested (Ti plants
Ti seeds
obtained)
Genotype
Phenotype
pFGC5941
27,500
(3) tsl-1/+
Wild-type
pFGCTSL
40,000
(28) +1+
Wild-type
Wild-type
Wild-type
Wild-type
Partial rescue of
tsl mutation.
Wild-type,
however half of
these plants
displayed a giant
phenotype
which did not
transmit to the
next generation.
Wild-type
(5) tsl-l/+
pFGCTLK
36,000
pFGCnTSL-cTLK
54,000
(1) +1+
(2) tsl- 11+
(1) tsl-1/tsl-1
pFGCnTSL-cTLK #16
(14) +1+
(3) tsl- 11+
115
Figure 29 Phenotype of the tsl-1 mutation (floral) and complementation of the
mutation with the pFGCnTSL-cTLK construct.
(A) Wild-type Arabidopsis thaliana flower. (B) Example of a tsl- 1 flower. Flowers
are missing organs, some organs may be misshapen and have an unfused gynoecium
and stigmatic tissues may generally be absent. The plants do not set seed at 22°C. (C)
and (D) examples of second generation of the pFGCnTSL-cTLK line segregating for
tsl- 1 but not pFGCnTSL-cTLK. Phenotype is same as tsl- 1. (E) and (F) examples of
a tsl-1 plant transformed with pFGCnTSL-cTLK (T2 generation). The mutant
phenotype is partially rescued, in that there is some stigmatic tissue present and seeds
are set at 22°C. Diagrams represent positions of floral organs; the outer half-ovals
represent sepals, the lines resent petals, the circles represent stamens and the
innermost set of half ovals represent the bicarpellate gynoecium.
116
Figure 30 Phenotype of the tsl-1 mutation (gynoecium) and complementation of the
mutation with the pFGCnTSL-cTLK construct.
(A) Wild-type Arabidopsis thaliana gynoecium. (B) Example of a tsl-1 gynoecium.
Gynoecium is unfused and stigmatic tissues is absent (C) and (D) examples of second
generation plants of the pFGCnTSL-cTLK line segregating for tsl-1 but not
pFGCnTSL-cTLK. Phenotype is same as tsl-1. (E), (F) and (G) examples of second
generation of a tsl-1 plant transformed with pFGCnTSL-cTLK. The mutant
phenotype is partially rescued. (E) and (F) are both partially unfused but have
stigmatic tissue. (G) is completely fused.
117
Rosette
Cauli ne
I
Figure 31
Phenotype of the tsl-1 mutation in Arabidopsis leaves and
complementation of the mutation by the pFGC nTSL-cTLK construct.
The third rosette (left) and the cauline leaves (right) are shown for segregating T2
generation plants. (A) Wild-type Arabidopsis thaliana. (B) tsl-1 mutation. (C) TslT1k16-2#55, segregating for tsl-1 only therefore the mutant phenotype is not rescued
and like in (B), the rosette leaves are more deeply serrated than in the wild-type. In
addition in (B) and (C), the cauline leaves curls around the new axis. (D) Tsl-Tlkl62#4, segregating for both tsl-1 and the transgene, pFGCnTSL-cTLK. The mutant leaf
phenotype is rescued, leaves are not deeply serrated and cauline leaves do not curl
around the new axis.
118
Of the 14 pFGCnTSL-cTLK Ti plants that had TSL/TSL genotype half
displayed a specific phenotype in that plants were bigger and a deeper green than
wild-type, rosette leaves were about twice as much as wild-type (15 rosette leaves to
about 7 rosette leaves in the wild-type) and were 2 weeks behind the wild-type plants
in flowering, that is a prolonged vegetative state. However, this phenotype was not
seen in T2 of the same lines.
35S: :TLK2 Transformants
Three transgenic lines corresponding to independent transformation events (Ti)
harboring the pFGCTLK construct were obtained. Of these lines, one had the +1+
genotype and two were +/tsl-1 (Table 8). The Ti plant pFGCTLK#2 (genotype +/tsl1) was allowed to self and set seed and the T2 progeny was analyzed as for the
pFGCnTSL-cTLK#16 T2 lines. Of the T2 progeny, 74% had the transgene in their
genome. This correlates to Mendelian inheritance of 3:1 indicative of one copy of the
transgene the original Ti plant. The genotype of pFGCTLK#2 T2 lines were 29%
wild-type, +1+, 53% heterozygous +/tsl-1 and 18% homozygous for the tsl-1 allele.
Of the plants that were homozygous for the tsl-1 allele, seven had the transgene
and were phenotypically rescued (Figure 32 through Figure 34 e.g. plant pFGCTLK#2
-19). Of the plants that were homozygous for the tsl-1 allele, two lacked the transgene
and displayed the tsl-1 phenotype (e.g. pFGCTLK#2-25).
119
Figure 32 Phenotype of the tsl-1 mutation (floral) and complementation of the
mutation with the maize TLK 2 gene.
(A) Wild-type Arabidopsis thaliana flower. (B) Example of a tsl-1 flower. Flowers
are missing organs, some organs may be misshapen and have an unfused gynoecium
and stigmatic tissue may generally be absent and does not set seed at 22°C. (C)
example of second generation plants of the pFGCTLK line segregating for tsl-1 but
not pFGCTLK. Phenotype is same as tsl-1, some stigmatic tissue present but
gynoecium is unfused with hole through its center. (D) example of second generation
plant of a tsl-1 plant transformed with pFGCTLK. The mutant phenotype has been
completely rescued by the introduction of the wild-type maize TLK 2 gene. Diagrams
represent positions of floral organs; the outer half-ovals represent sepals, the lines
resent petals, the circles represent stamens and the innermost set of half ovals
represent the bicarpellate gynoecium.
120
Figure 33 Phenotype of the tsl-1 mutation (gynoecium) and complementation of the
mutation with the maize TLK 2 gene.
(A) Wild-type Arabidopsis thaliana gynoecium. (B) Example of a tsl-1 gynoecium.
Gynoecium is unfused and stigmatic tissue is absent (C), (D) and (E) examples of the
pFGCTLK line segregating for tsl-] but not pFGCTLK (T2 generation). Phenotype is
same as tsl-1. (F) example of a tsl-1 plant transformed with pFGCTLK, T2
generation. The mutant phenotype is completely rescued.
121
Rosette
Cauline
Figure 34 Phenotype of the tsl-1 mutation in Arabidopsis leaves and
complementation of the mutation by the pFGCTLK construct.
The third rosette (left) and the cauline (right) leaves are shown for segregating
secondary transformed plants. (A) Wild-type Arabidopsis thaliana. (B) tsl-1 mutation.
(C) Tlk4-2#25, segregrating for tsl-1 only therefore the mutant phenotype is not rescued
and like in (B), the rosette leaves are more deeply serrated than in the wild-type. In
addition in (B) and (C), the cauline leaves curls around the new axis. (D) T1k4-2#19,
segregating for both tsl-1 and the transgene, pFGCTLK. The mutant leaf phenotype is
rescued, leaves are not deeply serrated and cauline leaves do not curl around the new
axis.
122
Table 9
Analysis of Transgenic Arabidopsis
T2
lines.
Number in brackets in the analyzed lines column refers to the actual number of plants
planted and initially analyzed. The first number refers to the number of plants that
were completely (genotyped for tsl-1, wild-type TSL and transgene and phenotyped)
analyzed.
Ti plant
Analyzed T2 Genotype
individuals
Transgene
present
Phenotype
PFGCnTSLcTLK#16
18 (55)
4 not present
tsl
14
Partial rescue of
tsl- 1: gynoecium
partially unfused
seed set, 4 sepals,
3-4 petals and
stamen, cauline
leaves uncurled,
rosette leaves are
wild-type
pFGCTLK#2
51(95)
tsl
Complete rescue
of tsl phenotype:
all plant parts are
wild-type.
tsl- 1/tsl- 1
(9 of 95)
2 not present
tsl- 1/tsl- 1
7
(15 ofSl)
2 not present
+1+
13
(27 of 51)
9 not present
tsl-l/+
18
Wild-type
Wild-type
Wild-type
Wild-type
123
Table 10
Organ Numbers in Transgenic Plants.
For each column, at least four flowers on each of 14 plants were analyzed except for
the wild-type control. For the control, ten flowers were examined.
Average #
Sepals in 56
flowers
Average # Average #
Petals in 56 Stamen in
flowers
56 flowers
Gynoecium
Stigmatic
tissue
WT Control
(untransformed)
4.0
4.0
6.0
fused in all
present
in all
pFGCTLK
(rescue)
4.0
4.0
6.0
fused in all
present
in all
pFCCnTSLcTCK
(partial rescue)
3.6±0.5
2.8±0.8
3.5±0.8
27 fused
8 almost fused
5 top split
present
in all
tsl-1
3.5±0.6
2±1.0
2.7+1.5
Not fused
11 tufts
29 absent
124
Discussion
The maize ZmTLK multigene family consists of two ancient
classes
The presence of three homologues of the Arabidopsis
TSL
protein in maize
raised questions concerning their origin and functional specialization. I have cloned
and analyzed the sequence structure of two of these genes namely, ZmTlkl and
ZmTlk2. I have evidence of a third homologue in the maize genome, ZmTlk3, from
partial genomic sequence analysis and mapping infonnation. It is, however, unclear at
this point whether ZmTlk3 is an expressed gene or a pseudogene since no ESTs (from
database searches) have been assigned as being positively ZmTlk3. On the other hand,
coding sequences of ZmTlk2 and ZmTlk3 are very similar (96% from 873 and 99%
from S60). Therefore, ESTs assigned as ZmTlk2 may represent both genes. In
addition, genomic sequences for the presumed 5' UTR and the first two exons of
ZmTlk3 (based on sequence similarity to ZmTlk2) were identified in GenBank
deposited as part of the maize sequencing project (accession numbers 42501291
and 45558003). Examination and comparision of these sequences to ZmTlkl and
ZmTlk2 showed that the presumed ZmTlk3 sequences appear to have a number of
errors in them in that within one exon there is a frameshift necessary to complete the
exon sequence. These errors may be sequencing errors as they are single base pairs
deletions/changes. Whether ZmTlk3 is an expressed gene or a pseudogene therefore
remains to be proven.
125
The ZmTlk genes are duplicated genes with very high sequence homology
both at the DNA and protein level. Sequence alignment of the ZmTlk genes over the
region for which we have good sequence for ZmTLK3 (last two exons and intervening
intron and 3 'UTR) indicate that ZmTlk2 and ZmTlk3 are more closely related to each
other than they are to ZmTlkl. Recently duplicated genes often retain close to or
above 90% sequence similarity between their introns, as has been shown for the maize
p1 and p2 genes (Zhang et al., 2000). The ZmTlk2 and ZmTlk3 genes, are 88%
identical in the thirteenth intron, 84% identical in the fifteenth intron and 94%
identical in their 3 'UTR sequences while their identity to ZmTlkl is 75% in all three
regions. This suggests that ZmTlk2 and ZmTlk3 are duplicate genes distinct from
ZmTlkl and can be considered to be one class of ZmTlk genes (ZmTlk2/3 class) with
ZmTlkl as a second separate class.
Maize and other Zea species arose from an allotetraploid thought to have
formed about 11 Myr ago. The ZmTlkl and ZmTlk2/3 classes appear to be ancient
orthologs from those progenitor species. ZmTlkl and ZmTlk2 share significant
homology over their coding regions and they have the same exons, which are identical
in size except for the first exon in which there is a deletion of four amino acids from
ZmTlkl. Homology between their introns is variable ranging from close to 80% to
none. Alignments of intron sequences that were close to 80% identical actually show
indels across the entire region and this is true of the 5'UTR sequence as well. The
observation that the non-coding sequences of ZmTlkl and ZmTlk2 are divergent is
consistent with there being two distinct classes of TLK genes in maize. ZmTlkl is
126
located on chromosome 1 L (position 1.12) and ZmTlk2/3 are on chromosomes 4c-L
(position 4.05) and SS (position 5.02) of the Pioneer composite map.
Maize is assumed to have been ultimately derived from teosinte. There are
five recognized species of teosinte: Zea diploperennis, Zea luxurians, Zea mays, Zea
nicaraguensis and Zea perennis. The species Zea mays is further divided into four
subspecies: ssp. huehuetenangensis, ssp. mays, ssp. mexicana, and ssp. parviglumis.
Zea mays ssp. mays, (maize or corn) is the only domesticated taxon in the genus Zea.
Phylo genetic reconstruction and progenitor inference studies studies show that the Zea
genus is a result of a hybridization between two progenitors (Gaut and Doebley, 1997
and references therein). This is the ancient segmental allotetraplodization event. Zea
mays ssp. parviglumis is then thought to have been domesticated to yield Zea mays
ssp. mays (Eyre-Walker et al., 1998 and references therein and Matsuoka, 2002).
With each progenitor providing a separate genome, it has lead to the prescence of
duplicated genes (on the duplicated chromsome segments) in the genome of mordem
maize and the other Zea grasses. The duplicated chromosomal segements are said to
be syntenic to one another. In maize, chromosome segments 1L and 5S are on one
such duplicated region. Since the most closely related ZmTlk genes (ZmTlk2 and
ZmTlk3) are not on the syntenic regions, it is likely that each ZmTlk class was derived
from the original progenitors that hybridized to yield the Zea species.
The sequence and mapping results lead to several hypotheses to explain the
existence of two genes in the ZmTlk2/3 class. Figure 5 lays out these hypotheses. The
first hypothesis is that each Zea progenitor had one TLK gene. This would mean that
ZmTlkl and one of the ZmTlk2/3 genes are of ancient origin and the other ZmTlk2/3
127
gene has risen as a result of a recent duplication of that class. This predicts that in the
Zea genus there will be two TLK genes with homology to the maize ZmTIk genes.
This hypothesis also predicts one TLK gene in grasses closely related to the Zea genus
that are not presumed to have been obtained from a similar hybridization event.
The second hypothesis is that there were in fact two TLK genes/maize
progenitor and after the hybrization event there were four TLK genes in the
allotetraploid. One of these genes could then have been lost via a deletion event
resulting in three copies of TLK genes in maize. All ZmTlk genes would therefore be
ancient genes. In this case, synteny between the ZmTlk genes cannot be predicted
since it is possible for all three genes to be in syntenous positions. This hypothesis
would predict that there are four or three TLK genes in two classes in the other Zea
species. If the maize progenitors diverged from the other grasses before the
duplication event that generated maize, then there could be any number of TLKs in
other grasses sampled.
There is a third possible hypothesis: there was one TLK originally in each
maize progenitor, however before the hybridization event to generate maize, a single
gene duplication occured in one progenitor (progenitor B) leading to two TLK genes
in progenitor B in non-syntenous positions and therefore there are three TLK genes in
maize. This hypothesis would predict three TLK genes in two classes (as in Zea
mays
ssp mays) in other members of the genus Zea. if this were the case, then, the
duplicated ZmTlk2/3 gene is also an ancient gene.
This study shows clearly that the ZmTlk2/3 class genes are not on syntenous
regions. It was also observed that, generally the wild members of the Zea genus have
128
three TLK genes in two classes, with one in the ZmTlkl class and two in the
ZmTlk2/3 class. Zea mays ssp. parviglumis, the supposed progenitor of maize has in
fact four TLK genes in two classes with two genes in each class. This may mean that
maize has lost one TLK gene. On the other hand it has been shown that Zea mays ssp.
parviglumis, generally has more sequence diversity than maize (Eyre-Walker et al.,
1998). In the Zea genus, the TLK genes could be divided into two classes based on
restriction enzyme digest patterns and sequence similarity in the twelfth intron. This
data supports the hypothesis that both classes of ZmTlk genes are ancient genes with
ZmTlkl arising from progenitor A and the ZmTlk2/3 genes coming from progenitor
B. The data obtained supports hypothesis two that each progenitor had two TLK
genes and there has been a subsequent loss of one TLK gene from some Zea genus
members.
Based on the sequence conservation in the ZmTlk2/3 class introns in Zea mays
ssp. mays, it would have been thought that ZmTlk3 is a more recent copy of ZmTlk2,
but with the data supporting the hypothesis that all three TLK genes existed prior to
hybridization, it can be concluded that those conserved untranslated sequences confer
some specificity of function in that class.
The grass genus most closely related to the Zea genus is Tripsacum.
Tripsacum dactyloides has at least four TLK genes in two separate classes similar to
the ZmTlk classes. This data may lend support to the second hypothesis for the
existence of three TLK genes in maize. Sorghum bicolor, the next close relative of the
Zea genus, has one TLK gene. The partial sequence obtained for the Tik gene from
Sorghum bicolor appears to be more closely related to ZmTlk2i3 than to ZmTlkl.
129
This would imply that the progenitor B that provided the ZmTlk2/3 class was more
similar to sorghum. Other more distantly related taxa like Hordeum vulgare (barley)
and Oryza sativa (rice) also have one TLK gene each. Avena sativa (oat) and Triticum
aestivum (wheat) both have at least two to three TLK genes. The Tik gene sequences
appear to be conserved in these grass species since primers from maize were used to
PCR amplify TLK from other grasses and to perform the limited sequence analysis.
The grass family shares extensive chromosomal synteny and grass consensus
maps have been generated based on 25 linkage groups of rice (Gale and Devos 1998).
Figure 35 shows circular maps representing the oat, Triticeace, maize, sorghum, sugar
cane, foxtail millet in relation to rice. The rice TLK gene is on chromosome 3. Rice
chromosome 3 shares synteny with maize chromosome segments 1L and 5S and with
sorghum chromosome C. This makes ZmTlkl and ZmTlk2/3 on 5S to be orthologous
to the rice Osllk. With the maize and rice TLK genes being on homoeologous
chromosomal segments, I can predict that the Sorghum bicolor TLK gene is on
chromosome C. There is however the caveat that while macrocolinearity (several
genes or map markers) is observed, microcolinearity (single gene loci) in the grass
genomes is not always the norm and there have been numerous exceptions as has been
shown for several orthologous genes between maize and sorghum, maize and rice and
sorghum and rice (Bennetzen, 2000 and Devos and Gale, 2000). This is due to
genome arrangements in the various grass species. Based on sequence similarity and
map positions, ZmTLK1, ZmTLK2, OsTLK and the sorghum TLK are orthologs.
130
GRASS
GENOMES
A
of.
FPJ
4
Maize
Sorghum
Cp
'
Sugar cane
Foxtail millet
B pt
\
'4
Rice
Ept
Ept
Figure 35 A Consensus grass comparative map (taken from Gale and Devos 1998).
131
Sequence and Phylogenetic analysis of the Plant and Animal
TLK proteins
Sequence and phylogenetic analysis of the ZmTlks indicate that they are
members of the well-conserved family of TOUSLED-like ser/thr protein kinases. The
TSL protein is a ser/thr kinase with a distinctive kinase catalytic domain at the Cterminal of the protein and an N-terminal regulatory domain. Kinases are classified
into families based primarily upon sequence similarity in the catalytic region and then
the classification is refined by extra catalytic region sequences and function. The
TOUSLED-like kinases are a model kinase family. Unique features of the TSL kinase
domain are shared by all 12 sequence-characterized TLKs, ZmTLKs included. In
subdomain I, TLKs have a GxGGFS motif (where x is K for all TLKs except the
mammalian ones in which it is R) instead of the canonical GxGxxG motif (Hanks and
Hunter, 1995). In subdomain II, the canonical AxK becomes ACK for all TLKs
except the mammalian ones in which it is AVK. Subdomain VIb has a HYDLKxxN
motif instead of HRDLKxxN. Subdomain VII becomes KVTDFG in plant TLKs and
KITDFG in metazoan TLKs conforming to KxxDFG. In subdomain VIII, the motif
GxxxxxApE becomes GTYWYLPPE in all TLKs while in subdomain IX KxxDfG is
DVWSAG in all TLKs. Such sequence conservation justifies the placement of these
proteins into the TOULSED-like kinase family and implies that these kinases will
share substrate homology.
The N-terminal region of TLK proteins are not sequence conserved; however
there is structure conservation in this region. The N-terminal of TSL has four nuclear
localization signals (NLS), three coiled-coil regions and a Q-rich region. Aside from
132
the Q-rich region in the N-terminal, all features of the TSL protein are shared by the
maize ILK proteins and are in the same relative positions in each protein. These
relationships between the ZmTLKs and TSL are shared by all TLKs characterized in
this study. Each TLK protein has at least two NLS and two to three coiled-coil
regions. DmTLK and AgTLK are predicted to have leucine zippers as well. TSL,
DmTLK and CeTLK are the only TLKs to have the Q-rich region. The function of the
Q-rich region is currently unknown; however it is interesting to note that with the
exception of rice, the TLKs that contain Q-rich regions are those that are known to be
single copy genes in their genomes. The Q-rich region may serve to allow for some
functional specialization.
To confirm the evolutionary relationship between ZmTLK1 and ZmTLK2 and
to explore their relationship to other TLK proteins, I performed a phylogenetic
analysis of TLK proteins from 12 species. The phylogenetic analysis of the TLK
genes was performed with three different methods. The UPGMA and neighbor joining
method gave the same tree. This tree is a measure of how each protein is related to the
next and it shows that the all the plant TLKs form one monophyletic group and the
animal ones form a separate group. The ZmTlk genes are more closely related to
TLKs of monocotyledonous origin than to Arabidopsis TSL which is a dicotyledonous
plant. ZmTLK1 forms a monophyletic group with OsTLK indicating a closer
relationship between ZmTLK1 and OsTLK than between ZmTLK2 and OsTLK. This
suggests that ZmTLK1 is the true ortholog of OsTLK in the maize genome.
Conservation of the TLK proteins across species implies that TLK activity is
highly likely to be conserved in all these different organisms and is likely to be
133
involved in a fundamental process. Since no TSL homologue was found in either
Saccharomycces cereviseae or Neurospora crassa, (two completely sequenced fungal
genomes) or in ESTs from other fungi (GenBank search June 2004), this process may
be related to some aspect of development unique to multicellular eukaryotes. It is also
possible that some other protein performs TLK functions in unicellular eukaryotes,
especially in light of the fact that the yeast Asfl has a stretch of acidic amino acids
that is not present in the Asfi genes from mammals, Drosophila and Arabidopsis.
ZmTLK2 is functionally equivalent to the Arabidopsis TSL
protein
The ZmTLK proteins being orthologs of TSL provided an opportunity to
examine the functional conservation of the ZmTLK enzymes. I have demonstrated
that ZmTLK2 is able to fully complement the tsl-1 mutant of Arabidopsis indicating
that ZmTLK2 (and by inference from the high homology between the ZmTlk genes,
ZmTLK1, as well) and TSL are functional homologs and operate in the same
pathways in maize and Arabidopsis respectively. This indicates that ZmTLK2 is a
fully functional kinase and is nuclearly localized.
The inability of the nTSL-cTLK hybrid construct to fbiiy complement the is!-]
mutation was unexpected and may be due to several reasons. One reason could be that
this is due to a position effect. The chromatin intergration site of the transgene has
led to expression in the plant being lower than needed to restore wild-type TSL
function. This effect can be verified in two different ways: 1) by examining the level
of expression of the nTSL-cTLK transgene from pFGCnTSL-cTLK#16 in the
134
transgenic plants as compared to the expression of the ZmTLK2 transgene in
transgenic plants and 2) since one transgene insertion was examined for each
construct, examination of a second independent nTSL-cTLK transgenic insertion
event may shed light on the possibility that the partial rescue shown by the
pFGCnTSL-cTLK#16 plant is due to position effects. It is also possible that the
hybrid protein was unable to fold properly and hence was not fully functional. The Cterminal of ZmTLK2 in the nTSL-cTLK construct may not have been folded properly
so that interaction partners presented to it by the TSL N-terminal region may have not
be fully held and phosphorylated and hence functionality would be lowered.
Biochemical studies in Arabidopsis suggest that TSL self-oligomerization is required
for catalytic activity (Roe at al., 1 997a). There is some evidence of oligomerization of
the human TLK proteins as well. If the nTSL-cTLK protein was unable to fold
properly, it would perhaps be unable to present quite the right surface for
oligomerization. Alternatively, it may be unable to form higher order oligomers if
more than dimerization is necessary for activity. Once oligomerization is impaired,
catalytic activity would be affected leading to a reduction in activity level of the
hybrid protein. In Drosophila, the level of DmTLK activity is involved in cell cycle
progression (Carrera et al., 2003) and it is possible that in Arabidopsis, reduced
activity of the hybrid protein is responsible for the partially rescued phenotype
observed. Another possibility is that the protein may not have been translated from
the first methionine but from an internal one leading to incomplete functionality of the
resultant protein.
135
The observed sequence and structure conservation in the TLK proteins point to
this class of kinases sharing an evolutionarily conserved role in both plant and animal
development and allows inferences to be made as to the ZmTLKs biological function
in maize. In addition to the strict conservation of the catalytic region sequence, each
TLK gene has a long N-terminal regulatory region that is structurally conserved across
both plant and animal kingdoms. This suggests that regulation of the TLK genes
andlor interaction partners and kinase substrates and hence pathways they are involved
in are similar. This hypothesis is supported by the results of investigation of these
genes in humans, Arabidopsis, C. elegans and Drosophila (Sillje' et al., 1999, Roe et
al., 1997a, Hans et al., 2003 and Carrera et al., 2003). In all of these organisms, TLK
proteins were found to be localized in the nucleus. Based on these facts, it is entirely
likely that the ZmTLK proteins are also nuclear localized. TLK proteins (humans,
Drosophila and Arabidopsis) have been demonstrated to interact with ASF1, (Silije' et
al., 1999, Carrera et al., 2003 and Ehsan et al., 2004) a chaperone for histones H3 and
H4. In Drosophila, this interaction was shown to be involved in chromatin assembly
during DNA replication and in fact TLK mutant cells halt in S-phase of the cell cycle
followed by apoptosis. In human cells, TLK activity is linked to DNA replication
during S-phase. A splice variant of HsTLK1 has also been shown to phosphorylate
histone H3 (Li et al., 1999). CeTLK and DmTLK do not phosphorylate histone H3
(Hans et al., 2003 and Carrera et al., 2003) however, in TLK mutants of Drosophila,
histone H3 levels and phosphorylation are reduced. Based on these findings, it is
established that TLK genes are involved in DNA replication, chromatin assembly and
progression through S-phase of the cell cycle.
136
There are additional levels of control of the TLK proteins since the conservation
in the N-terminal region is structural not amino acid specific. This may be important
for specific protein interaction binding specificity and may also lead to specific
functions for a TLK protein in addition to a general role in chromatin assembly during
S-phase. This hypothesis has been supported by the finding that in Arabidopsis, TSL
interacts with ASF1 via its C-terminal and with TKII, a myb-SANT protein via its N-
terminal region (Ehsan et al., 2004). TKII, being a myb-SANT protein is likely to be
a transcription regulating protein. In C.
elegans,
mutations in CeTLK indicate a role
for CeTLK in transcription since mutations causing CeTLK activity loss lead to a
phenotype reminiscent of C. elegans transcription mutants. Indeed, the levels of
embryonic RNAPII transcription dependent proteins are shown to be affected by
CeTLK loss of function mutations (Hans et al., 2003).
ZmTlk genes are differentially expressed
Duplicated genes often have very different evolutionary fates. The differential
expression pattern of the maize TLK genes suggest that they may have overlapping
roles in some pathway and have nonredundant functions that have become specialized
during maize evolution. All Tlk genes studied to date have been demonstrated to be
ubiquitously and constitutively expressed with varying levels of expression in specific
tissues. Results from Arabidopsis and mice/human expression analysis indicate that
TLK expression is higher in dividing tissues (Roe et al., 1993, Sillje'
et al.,
1999 and
Ebsan et al., 2004). This study supports these findings, though there are some
differences in the expression pattern of ZmTlk genes as compared to that of
137
Arabidopsis,
humans and mice. ZmTlk2 and ZmTlk3 are duplicated genes with
extremely high sequence homology. mRNA sequences for these two genes may
therefore be very closely related. I have assumed that may be the case and hence I
analyzed the expression data I obtained as for ZmTlk2/3 and ZmTlkl. It is possible
though that ZmTlk3 is a pseudogene since I have been unable to positively identify
EST entries for this gene in GenBank. This may be the case since in the evolution of
duplicated genes, one of the versions may be lost (Conery and Lynch, 2000).
The maize root and young leaf show spatial separation of cell proliferation and
expansion. Cell division is high at the meristematic zone, i.e. root tip and section of
leaf closest to the sheath, while expansion occurs progressively along the length of the
root and leaf. ZmTlk expression is higher at the root tip and dividing region of the
leaf as compared to expanding root or leaf sections. ZmTlk transcripts accumulate to
a higher level in root tissue than in leaf tissue and this is true for both dividing and
expanding tissues. In particular, ZmTlk2/3 expression levels in the expanding tissues
of the root are higher than ZmTlkI expression indicating a tissue specific or functional
divergence role for ZmTlk2/3 in this tissue. This implies that these genes are indeed
associated with cell division but have a role in cell expansion or differentiation as
well.
While HsTLK activity is directly related to DNA replication, HsTlk expression
could not be correlated to mitotic activity (Sillje et al., 1999). In maize, I have shown
that ZmTlk expression correlates more with S-phase of the cell cycle in contrast to the
expression of CycZmel which is correlated with M-phase activity of maize tissues.
138
Endoreduplication is a wide-spread phenomenon in both plants and animals. In
Arabidopsis, endoreduplication occurs in vegetative tissues e.g. stem but not the
inflorescences (Gaibraith et al., 1991). Arabidopsis TSL expression is highest in the
floral buds and lowest in the stem (Roe et al., 1993). In maize, I have shown that
while there is a higher level of ZmTlk expression in immature floral tissue (as in
Arabidopsis), the highest expression of the ZmTlk2/3 class in particular is found in the
developing endosperm which is undergoing endoreduplication. Endoreduplication
(chromosome replication in the absence of nuclear division) is the means by which
nuclear polyploidization occurs most frequently in metabolically active tissues. In the
developing maize endosperm, the triploid nucleus undergoes multiple synchronous
cell cycles without cytokinesis during the first three days to form a syncytium. After
the syncytium is cellularized, the endosperm undergoes several rounds of a regular
mitotic cell cycle giving rise to several 100 nuclei up till about 9 - 12 days after
pollination (DAP) after which it switches to an endoreduplication cycle consisting of
repeated cycles of S-phase followed by a short gap phase (Larkins, 2001).
Endoreduplication continues until about 21 27 DAP (Young et al., 1997 and Dukes
et al.,
2002) after which the central cells of the endosperm begin to undergo cell death.
The length of time and extent of endoreduplication varies for maize inbred lines
(Cavallini
et al.,
1995 and Dukes et
al.,
2002) and affects the amount of storage
protein (zeins) the endosperm accumulates. Endoreduplication in the maize
endosperm is coincident with zein production and with auxin increase/ cytokinin
decrease in the endosperm (Lur and Setter, 1993). Endoreduplication is thought to be
a means of increasing the cell volume. It is also thought to provide templates for more
139
gene expression to accommodate the need for higher transcripts levels in a
differentiating tissue.
ZmTlk2/3 expression increases significantly after 9-DAP and the expression
level is highest in 20-DAP endosperm. The timing of this increase in expression is
coincident with the endoreduplicating cycle progression. The increase in ZmTlk2/3
expression is not accompanied by a concurrent increase in ZmTlkl expression.
ZmTlk2/3 levels in endo sperm are six times higher than ZmTlkl levels in other maize
tissues. This suggests that ZmTlk2/3 has a specific role in the endosperm in addition
to its general role in chromatin assembly during DNA replication. This role may be
distinct from the chromatin assembly role since ZmTlk2/3 levels are much higher than
the levels of histone H3 observed in the same tissues. It must be noted that there are at
least 30 variants of histone H3 in the maize genome and hence the level of bistone H3
reported here may not actually reflect the complete picture of histone H3 levels in
these tissues. However, the PCR primers used for investigating the histone 113
expression were obtained by aligning a number of histone H3 sequences and choosing
a conserved region for the primer generation. It is important to note that histone H3
levels are comparable with the ZmTlkl expression levels and are significantly higher
than the cyclin B 1 levels. Cyclin B 1 levels have been previously noted to decrease
upon entry into endoreduplication (Sun et al., 1999). This suggests that ZmTlkl may
be the Tik gene involved in chromatin assembly in the endosperm.
ZmTlk2/3 may have a role in transcription in addition to the role in replication
as found for the CeTLK protein. This would explain the
high
level of expression in
140
ZmTlk2/3 in the expanding region of the root and in the endosperm tissues since
transcription of zeins in the endosperm is very high at this time. It is also possible that
in fact, the high levels of ZmTLK2/3 in the endoreduplicating endosperm tissues are
part of a regulation pathway that coordinates and leads to the down-regulation of
endoreduplication as its levels increases as endoreduplication time increases, perhaps
leading to cell death and endosperm maturation.
Conclusions and Recommendations
The ZmTlk genes are functional homologs of Arabidopsis TSL. The expression
patterns of the genes are consistent with involvement in chromatin assembly during
DNA replication, cell cycle progression and development of the plant, as has been
shown in animals. The ZmTLK proteins have overlapping expression patterns in
most tissues of the maize plant, however the duplicated ZmTLK2/3 class is likely to
have another role divergent from ZmTLK1 in the maize root and endospenn. The
conservation of structure indicates similar major functions of all the ZmTLK genes but
their distinct temporal pattern of expression in the endosperm indicates regulation of a
specific pathway as does their spatial pattern of expression in the root.
In order to determine the role that ZmTlk2 is likely playing in the endosperm,
the protein level and phosphorylation state of ZmTlk2 must be determined during
endosperm development. It will also be necessary to determine and correlate the
141
actual amount of time the maize inbred line W22 endosperm is undergoing
endoreduplication, the level of endoreduplication and ZmTLK2 activity levels. In
addition, it must be investigated as to whether ZmTlk2 has different protein
interactions or substrates in the endosperm as compared to other maize tissues.
Phenotypic examination and biochemical analysis of maize endosperms of plants with
mutations in ZmTLK2/3 would be instrumental in investigating the function of the
ZmTlk2/3 genes in the endoreduplicating endosperm.
Since the sequence of ZmTlkl and ZmTlk2 are now completely known, it
should be possible to obtain maize mutants for both of these genes by screening the
Pioneer TUSC library or by using other methods such as RNA interference.
142
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