AN ABSTRACT OF THE THESIS OF
Yeoniin Kim Veach for the degree of Master of Science in Plant Physiology
presented on June 12, 2002.
Title: CYTOKININ 0-GLYCOSYLTRANSFERASES: BIOCHEMICAL
CHARACTERISTICS IN VITRO AND DEVELOPMENTAL REGULATION IN
TRANSGENIC PLANTS.
Abstract approved:
Redacted for privacy
Machteld C. Mok
Cytokinins are plant hormones regulating cell division and differentiation.
Most developmental events from germination, vegetative morphogenesis to seed
and fruit set are influenced by cytokinins. t,ns-Zeatin is the major natural
occurring cytokinin. Its isomer, cis-zeatin, and its derivatives are usually present in
low quantities and have traditionally been considered as adjunct components due
to their low biological activity. Glycosylation of cytokinins occurs frequently and 0-
glycosylation genes of t,ns-zeatin have been cloned in our lab. Determining the
function of 0-glycosides and the regulation of gene expression are part of a larger
on-going project.
Current literature indicates that cis-zeatin derivatives occur as predominant
cytokinins in specific organs of certain plant species, such as chickpea seeds,
potato tubers and rice roots. These observations, together with the recent cloning
of maize genes encoding 0-glucosyltransferses preferring cis-zeatin to transzeatin, suggest that cis-zeatin may have a more important role than currently
recognized. Within the long-term objective of understanding the possible role of
cis-zeatin in plant development and the function and regulation of cytokinin 0-
glycosylation, this study was undertaken to contribute to two specific areas, the
characterization of two cis-zeatin 0-glucosyltransferases (cisZOGl and cisZOG2)
and determining promoter activity of a t,ans-zeatin O-xylosyltransferase gene
(ZOXI) in transgenic plants.
Open reading frames (ORFs) of the cisZOGl and cisZOG2 were cloned
into expression vectors and large quantity of recombinant enzymes was obtained.
Substrate specificity and reaction properties of cisZOGl and cisZOG2 were
examined. The optimal pH of the enzymatic reaction is about 7.5. The Km for ciszeatin is about 46i.LM for cisZOGl and 961.iM for cisZOG2. UDP-glucose (UDP-
Glc), the sugar donor, has KmS of 0.1 and 0.59mM for cisZOGl and cisZOG2,
respectively. The Km values were within the expected range of cytokinin
metabolism enzymes. The affinity of these enzymes to cis-zeatin was 11 fold
higher than tins-zeatin. Competition experiments with other cytokinins indicated
that cis-zeatin riboside, and thidiazuron are strong inhibitors while t,ns-zeatin,
kinetin and N6-benzyladenine are slightly inhibitory. These findings, combined with
detecting substantial amounts of cis-zeatin derivatives in all tissues, suggest
specific metabolic pathways for cis-zeatin in maize.
The regulation of trans-zeatin 0-glycosylation gene expression was
studied using an available sequence of ZOXI (zeatin O-xylosyltransferase)
containing the promoter fused with the reporter gene GUS (p-glucuronidase).
Transgenic tobacco and Arabidopsis containing the pZOXI::GUS and control
constructs (superpromoter::GUS and no promoter::GUS) were generated.
Reporter gene activity mediated by pZOX1 was observed in immature seeds of
transgenic tobacco as expected of the promoter in its native host, Phaseolus. In
vegetative tissues, pZOX1 mediates gene expression only in trichomes and stem
hairs. As the formation of these tissues from epidermal cells involves a switch in
cell division plane as well as endoduplication, the expression of zeatin 0glycosylation genes in native hosts may be related to transient reduction of active
cytokinins to accommodate alternating cydes of rapid duplication and rest. The
absence of pZOXI::GUS activity in transgenic Arabidopsis may indicate a
difference in required transcription factors between this model plant and tobacco
and legumes. The results and plant materials will serve as useful information and
materials to further explore the induction of gene expression by hormones and
environmental cues.
CYTOKININ 0-GLYCOSYLTRANSFERASES:
BIOCHEMICAL CHARACTERISTICS IN VITRO AND
DEVELOPMENTAL REGULATION IN TRANSGENIC PLANTS
by
Yeonjin Kim Veach
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Master of Science
Presented June 12, 2002
Commencement June 2003
Master of Science thesis of Yeonjin Kim Veach presented on June 12. 2002.
APPROVED:
Redacted for privacy
Major Professor, representing Plant Physiology
Redacted for privacy
Director of Plant Physiology Program
Redacted for privacy
Dean of Grajiiatë School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to
any reader upon request.
Redacted for privacy
eonjin Kim Veach, Author
ACKNOWLEDGEMENTS
I would like to express my deep appreciation to Professor Machteld Mok
who supported me throughout the course of my research. Her encouragement will
remain with me wherever I go. I also want to thank Professor David Mok for
assisting me through his knowledge and excellent microscopy skill. I sincerely
thank Ruth Martin for providing me guidance dunng my two years of study and for
sharing friendship with my family and me. I would also like to thank Professor Pat
.Breen and Professor Alan Bakalinsky for serving as members of my committee. I
would like to thank Professor Michael Schimerlik for letting me use Scientist
Micromath program and for his kindness. I would also want to thank Professor
Sang-Jun Kang in Korea who encouraged me to continue my education in
graduate school and assisted me with my admission to OSU.
Finally, I deeply thank my family both here and in Korea for the unlimited
support they have given me the past two years. Especially, my husband Tim and
my son Rahim encouraged and supported me throughout this big step of my life,
and I would like to thank them with all my heart.
TABLE OF CONTENTS
Page
1. INTRODUCTION AND LITERATURE REVIEW ..............................
i
Introduction to cytokinins ....................................................
1
Natural cytokinin metabolites and their function ........................
3
Cytokinin biosynthesis .........................................................
6
Enzymes and genes involved in cytokinin metabolism ...............
8
Objectives of this study .......................................................
12
.......................................................................
14
References
2. BIOCHEMICAL CHARACTERIZATION OF cis-ZEATIN 0GLUCOSYLTRANSFERASE GENES, cisZOGl AND cisZOG2
.........
19
Abstract...........................................................................
19
Introduction.......................................................................
20
Materials and Methods .........................................................
21
Generation of recombinant Protein ...............................
Biochemical charactenzation of recombinant enzymes
21
Results............................................................................
23
Description of cisZOGl and cisZOG2 ...........................
Biochemical characterization of cisZOGl and cisZOG2
23
24
22
Discussion.........................................................................
30
References.......................................................................
34
TABLE OF CONTENTS (Continued)
Page
3. PROMOTER STUDY OF A GENE ENCODING A tmns-ZEATIN 0GLYCOSYLTRANSFERASE ......................................................
37
Abstract...........................................................................
37
Introduction......................................................................
38
Materialsand Methods .......................................................
39
Transformation vectors .............................................
Tobacco transformation ............................................
Arabidopsis transformation ........................................
PCR and Southern analysis .......................................
39
40
41
Induction conditions .................................................
42
43
45
Results............................................................................
46
Genetic characterization of transformants .....................
GUS expression of transgenic plants ...........................
Induction of pZOX1 promoter by external factors ............
46
49
52
Discussion.......................................................................
53
References......................................................................
57
.......................................................
58
BIBLOGRAPHY......................................................................
59
GUSassay .............................................................
4. GENERAL CONCLUSION
LIST OF FIGURES
Figure
Page
1.1. Naturally occurring cytokinin metabolites
and kinetin (first identified cytokinin by Skoog group) .....................
4
2.1. Comparison of the sequences of cisZOGl and cisZOG2 genes .......
24
2.2. Diagrammatic presentation of the upstream sequences .................
24
2.3. Separation by HPLC of the substrate (cis-[3H]zeatin) and product
(0-glucosyl cis-[3Hjzeatin) obtained from the cisZOG enzyme
reaction...............................................................................
26
2.4. The effect of the pH on the conversion of cis-[3H]zeatin to
cis-[3H]zeatin O-glucoside by cisZOGl (A) and cisZOG2(B) ............
26
2.5. The effect of substrate concentration on the velocity of
glucosyl transfer by cisZOGl and cisZOG2 .................................
27
2.6. Cytokinins as competitors of the conversion of cis-[31-1]zeatin
to its glucoside by cisZOGl ....................................................
29
3.1. Diagrammatic illustration of tobacco samples used for GUS assay
A. Leaf and stem samples ........................................................
B. Stages of tobacco flowers from very ung buds (1)
toseed pods (7) .................................................................
3.2. PCR products obtained from transgenic tobacco using primers
based on the GUS gene sequence ............................................
44
44
48
3.3. Southern blots of transgenic tobacco and Arabidopsis
probedwith GUS ...................................................................
49
3.4. Young seeds of pZOX1::GUS tobacco expressing GUS .................
51
3.5. Transgenic tobacco leaf tnchomes expressing GUS ......................
51
3.6. Hairs on stems of pZOX1::GUS tobacco showing GUS activity .......
52
LIST OF TABLES
Page
Table
of cisZOGl and cisZOG2 .........................................
28
3.1. Segregation of T1 transgenic tobacco for kanamycin resistance .........
47
3.2. Segregation of T1 transgenic Arabidopsis for kanamycin resistance ...
48
2.1. Km and
Vmax
CYTOKININ 0-GLYCOSYLTRANSFERASES:
BIOCHEMICAL CHARACTERISTICS IN VITRO AND
DEVELOPMENTAL REGULATION IN TRANSGENIC PLANTS
CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
Introduction to cytokinins
Cytokinins are an important group of plant hormones. The name is derived
from the fact that they promote cell division, or cytokinesis. The first cytokinin was
identified by Skoog, Miller, and colleagues in 1955 as a factor stimulating growth of
tobacco pith tissue and was named kinetin (Miller et al., 1955). Since that time,
cytokinins have been isolated from a wide variety of different plants (Mok and Mok,
1994).
Cytokinins exist in plant cells as free molecules or incorporated into tRNA
(Skoog and Armstrong, 1970). The most important free form of cytokinin is zeatin
(trans-zeatin), but many derivatives of zeatin as well as related compounds are
also known to occur. cis-Zeatin is the major form of cytokinin present in tRNA.
This cytokinin can be found at the 3' end of the anticodon sequence of certain
tRNAs and it alters the shape of the tRNA molecule. It is believed that the
incorporated cytokinin plays a role in the interaction of the anticodon loop with
mRNA-binding sites, thereby influencing mRNA translation (Skoog and Armstrong,
1970). However, it is the free cytokinins that participate in regulating cell division
and plant development.
Cytokinins have diverse effects on plant development. The dassical
studies by Skoog and Miller (Skoog et al., 1957) showed the prominent effects of
kinetin on bud formation in tobacco callus cultures. Since that time, studies using
numerous other plants have confirmed the morphogenic effects of exogenous
cytokinins (Mok and Mok, 1994). In addition to the effects on de novo bud and
shoot formation, cytokinins stimulate the growth of shoots from already formed
buds. This has been shown by application of exogenous cytokinins (Skoog and
Miller, 1957) as well as changes in the levels of endogenous cytokinins through
genetic modifications. For example, plants containing the isopentenyltransferase
(ipt) gene from Agrobactenum fume faciens, causing elevated cytokinin
biosynthesis, have generally increased lateral branching (Smigocki and Owens,
1988; SchmUlling et al., 1989). The high level of endogenous cytokinins in the
supershoof mutant of Arabidopsis showed an increased number of menstems in
leaf axils and increased branching (Tantikanja et al., 2001). Conversely,
decreasing cytokinin levels by over-expression of cytokinin oxidase, an enzyme
inactivating cytokinins, in transgenic tobacco resulted in smaller apical meristems
and stunted shoots (Werner et al., 2001).
Cytokinins also play a role in delaying senescence of plant leaves. This
was first reported by Richmond and Lang (1957), who showed that application of
kinetin could lead to chlorophyll retention in Xanthium leaves. More recently, Gan
and Amasino (1996) demonstrated the pronounced effects of endogenous
cytokinins in delaying senescence by transforming plants with the ipt gene driven
3
by a senescence-induced promoter. Cytokinins also have the ability to increase
chlorophyll in non-senescing leaves, which seems to be due to differentiation of
plastids (Van Staden et al., 1988). However, the delay of senescence by
cytokinins is thought to be associated with a decrease of protein and membrane
degradation (Thimann, 1987; Letham, 1988). Application of cytokinins prevents
formation of free radicals and lowers lipases and lipoxigenases involved in
degradation of lipids in the membrane (Thomson et al., 1987; Letham, 1988).
Exogenous cytokinins also have clear effects on leaf expansion (Nielsen
and Ulvskov, 1992). This may be linked to the ability of cytokinins to enhance sink
strength for allocation of nutrients (Gersani and Kende, 1982; Guivarc'h et al.,
2002). Cytokinins also promote seed germination and are involved in regulation of
stomatal opening, development of nodules, nutrient availability, and flowering (Mok
and Mok, 1994). However, other plant hormones are important in these as well.
Natural cytokinin metabolites and their function
Natural cytokinins have either an isoprenoid or an aromatic side chain at
the N6-position of adenine. Cytokinins with an unsaturated isoprenoid side chain
are most common, of which trans-zeatin (Fig. 1.1) is considered the most important
due to its high activity and ubiquitous occurrence in higher plants. Other isoprenoid
cytokinins
include
cis-zeatin,
N6-(\2-isopentenyl)adenine
(i6Ade),
and
dihydrozeatin, the latter having a saturated side chain (Fig.1.1). N6-benzyladenine
(BAP) and its hydroxylated derivatives, the topolins, are naturally occurring
4
cytokinins with nng substitutions at the N6-position (Stmad, 1997). Although kinetin
was first identified in natural extract (Miller et al., 1955), it seems to be a product of
autoclaving DNA and not a natural free cytokinin.
OH
OH
OH
HN
HN
NyN
trans-zeatin
cis-zeatin
NN
dihydrozeatin
HNQHN
NLN
N'LrN
LNJN
N6-(i2-isopentenyl)adenine
H
N6-benzyladenine
kinetin
i6Ade
Figure 1.1. Naturally occurring cytokinin metabolites and kinetin
(first identified cytokinin by Skoog group).
Since the naturally occurring cytokinins are adenine derivatives, they
undergo some metabolic conversions in common with adenine. Therefore, plant
tissues commonly contain the free base forms together with the corresponding
ribosides and nucleotides. Furthermore, the adenine ring can be glucosylated at
5
the 3-, 7- and 9-positions. Glycosytation of the hydroxylated side chain occurs
frequently, mainly 0-glucosylation, although O-xylosylation is also known to occur
(Lee et at., 1985; Mok and Mok, 1987). 0-acetylzeatin and 9alanylzeatin (lupinic
acid) have been identified in some plants (Letham and Zhang, 1989; Stmad, 1997;
Vañková, 1999).
The various cytokinin metabolites may have different functions.
It is
generally assumed that the free base is the active form of cytokinin. Of these,
tmns-zeatin is usually the most active in bioassays, while i6Ade, dihydrozeatin and
BAP have somewhat lower activity. cis-Zeatin is only weakly active and its role is
unclear. It possibly could serve as a precursor to t,ns-zeatin since cis-trans
isomerization can take place (Bassil et aL, 1993).
Other metabolites may have some specialized function. Cytokinins are
synthesized in root menstems and passively transported from roots to other
organs through the xylem. The nucleotides may be the transport forms since the
cytokinins in the xylem are predominantly present as nucleotides (Letham, 1994).
0-Glycosides are considered to be reversible storage products since they
can readily be converted back to the aglycones by f-gIucosidases. Moreover, 0glycosylation can provide resistance to attack by cytokinin oxidases, which cleave
unsaturated isoprenoid side chains. In contrast, the N-glucosylation at the 7- and
9-position of the adenine ring is irreversible, and N-glucosides display no activity in
bioassays (Lethem et al., 1983). N-Glucosylation at the 3-position is reversible
(Letham et at., 1975), but it may not protect against degradation by cytokinin
oxidases (Armstrong, 1994).
Cytokinin biosynthesis
There are two possible cytokinin biosynthetic pathways. One is the de
novo (direct) biosynthetic pathway and the other is indirect cytokinin formation by
breakdown of tRNA. In the de novo pathway, condensation of
dimethylallylpyrophosphate (DMAPP) and 5'-AMP is mediated by
isopentenyltransferase (DMAPP: AMP transferase) and
N6-
(A2-isopentenyl)
adenosine 5'-phosphate (i6AMP) is formed. This ribotide can subsequently be
converted to the riboside and free base. In the indirect pathway, cytokinins (ciszeatin and i6Ade) occuning adjacent to the anticodon of tRNA can be released
during tRNA breakdown. However, turn-over of tRNA is not sufficient to account
for the relatively high levels of free cytokinins in many plant tissues.
The first isopentenyl transferase (ipt) was partially purified from
Dictyostelium (Taya et al., 1978). Since that time, several cytokinin biosynthetic
genes have been identified, first in bacteria nd recently also in plants. The ipt (or
tmr) gene of Agrobactenum tumofaciens, one of the genes responsible for tumor
growth in plants, encodes a 27 kD protein that has isopentenyltransferase activity
(Barry et al., 1984). This gene has an eukaryotic promoter and is thus not
expressed in Agrobacterium but only in the host plant (Buchmann et al., 1985).
The tzs gene of Agrobaterium, located on the Ti plasmid but outside the T-region,
has high homology to the ipt gene and also encodes an isopentenyl transferase
(Kaiss-Chapman et aL, 1977). The miaA gene of Agmbacterium is also highly
homologous to the ipt gene and encodes a DMAPP: tRNA isopentenyltransferase
(Gray etal, 1992).
Recently, significant progress was made in identifying cytokinin
biosynthetic genes of higher plants. In 2001, nine ipt homologs of Arabidopsis
thallana were characterized after the Arabidopsis genome was sequenced (Takei
et al., 2001; Kakimoto et al., 2001). While At IPT2 and AtIPT9 encode putative
DMAPP:tRNA transferases, the other AtIPT genes encode other enzymes involved
in biosynthesis of free cytokinins (Kakimoto, 2001; Takei et aL, 2001).
Interestingly, AtIPT4 uses AlP or ADP as a substrate instead of AMP (Kakimoto,
2001). The Sho (Shooting) gene of Petunia hybrida shows homology to the
Arabidopsis ipt genes (Zubko et al., 2002) and transgenic petunia plants
overexpressing Sho accumulated isopentenyl-type cytokinins.
Plant tissues contain primarily zeatin and its derivatives. The enzymes
described above mediate formation of isopentenyl-type cytokinins. Logically,
hydroxylation of i6Ade (or i6Ado, i6AMP) to zeatin (or zeatin riboside, ribotide)
would be the next step in the biosynthetic pathway. Indeed, ipt-containing bacteria
were found to secrete zeatin (Davies, 1995)and also plant tissue transformed with
the ipt gene accumulated zeatin, zeatin riboside, and the glucoside. In support of
this contention, hydroxylation of i6Ade was observed in some species such as
Actinidia (Einset, 1984). However, in other plant species direct convertion of i6Ade
to zeatin was not observed even though zeatin accumulates in them (Mok and
Mok, 2001). Lately, indirect evidence was provided suggesting direct biosynthesis
of zeatin-type cytokinins, without isopentenyl intermediates (Astot et al., 2000). It is
possible that some isopentenyl transferases can transfer a hydroxylated side
chain, in addition to the isopentenyl side chain. However, occurrence of precursors
with hydroxylated isoprenoid side chains has not been reported. The unavailability
of synthetic compounds with similar configuration also hampers future research.
Enzymes and genes involved in cytokinin metabolism
The major enzymes and corresponding genes mediating cytokinin
metabolism have been described by Mok and Mok (2001). The enzymes include
zeatin 0-glucosyltransferase, zeatin O-xylosyltransferase, cis-zeatin 0glucosyltransferase, N-glucosyltransferase, -glucosidase, cytokinin oxidase, N-
alanine transferase, cis-trans isomerase, and zeatin reductase.
Zeatin 0-glucosyltransferase was first isolated from lima bean (Phaseolus
lunatus) seed (Dixon et al., 1989). This enzyme uses UDP-Glc as the preferred
sugar donor substrate (Km 0.2mM) to form 0-glucosyizeatin, but it can also use
UDP-Xyl (Km 2.7mM) to form 0-xylosylzeatir%. The gene encoding this enzyme,
ZOG1 (for zeatin O-glucosyltransferase), was isolated by Martin et al. (1999a) by
screening a P. Iunatus expression library with monoclonal antibodies specific to
the enzyme. The open reading frame (ORF) is 1380 nucleotides long and encodes
a 51 kD protein. There is no intron in the ZOGI gene (Martin et al., 1999a).
Zeatin O-xylosyltransferase occurs in seeds of the common bean (P.
vulgaris). It is very similar to the zeatin 0-glucosyltransferase of P. lunatus but
uses UDP-Xyl as sugar substrate and not UDP-Glc (Turner et al., 1987).
The zeatin O-xylosyltransferase (ZOXI) gene was isolated from P. vulgaris by PCR
using primers based on the ZOGI sequence (Martin et at., 1999b). The ORF of
ZOX1 has 1362 bp and encodes a 51 kD peptide.
The ZOXI and ZOG1 genes are very similar in DNA (93% identity) and in amino
acid sequence (90% identity).
A cis-zeatin 0-glucosyltransferase gene was isolated from maize based on
expressed sequence tags (ESTs) with high homology to ZOG1 (Martin et at.,
2001). The gene has a 1401 nucleotide-long ORF, encoding a protein of 51.1 kD
with 60% identity to the Phaseolus ZOG1 at the DNA level and 41% at the protein
level (Martin et al., 2001). This gene, like the other cytokinin glycosyltransferase
genes, does not have any intron. The cisZOGl enzyme uses cis-zeatin and UDP-
GIc but not UDP-Xyl to form cis-zeatin 0-glucoside. Expression of the cisZOGl
gene as determined by molecular beacons, was found to be highest in roots,
followed by cobs and kernels, with low in leaves (Martin et al., 2001). The
occurrance of cis- specific enzyme and the identification of cis-zeatin in many plant
species (reviewed by Mok and Mok, 2001) suggest that there might be cis-zeatin
specific metabolic pathways in plants and that cis-zeatin and its glucosides may
play a role in cytokinin homeostasis (Martin et al., 2001).
In addition to the 0-glucosyltransferases, an enzyme converting zeatin to
its N7- and N9-glucosides has been idetified (Entsch and Letham, 1979; Entsch et
al., 1979). This enzyme recognizes a number of substrates, including adenine, but
conversion is higher with cytokinins (Entsch et al., 1979). The Km for zeatin is 150
tM and the main product is the N7-glucoside. The enzyme has a mass of about
46.5 kD. Genes encoding N-glucosyltransferases have not been isolated.
10
Cytokinin activity of 0-glucosyizeatin can be restored by p-g(ucosidase
cleaving the glucose moiety. A maize p-glucosidase capable of cleaving the
glucose of 0-glucosylzeatin was partially purified as a 60 kD protein (Campos et
al. 1992). Isolation of the gene encoding this enzyme, Zm-p60. I (or glul), has
been reported (Brzobohat et al, 1993). This gene is about 5 kb and consists of 12
exons and 11 introns. The recombinant enzyme cleaves 0-glucosyizeatin and
kinetin-N3-glucoside and several other artificial and natural substrates, but not the
N7- and N9-glucosides. Due to this wide substrate recognition, hydrolysis of
glucosides may not be as precisely regulated as 0-glucosylation (Mok and Mok,
2001).
Cytokinin oxidases convert cytokinins with unsaturated isoprenoid side
chains such as zeatin, zeatin riboside, and i6Ade to adenine or adenosine. The
role of this type of enzyme is to decrease active cytokinin, and to prevent
accumulation of toxic levels of the hormone (Mok and Mok, 1994). Since the
isolation of the first cytokinin oxidase from kbacco callus tissues 30 years ago
(Paces etal., 1971), this type of enzyme has been reported in maize, beans,
poplar, wheat, and Vinca rosea crown gall tissues (Armstrong, 1994).
A maize cytokinin oxidase gene, ckxl or CKO, was cloned independently
by two groups (Houba-Herin et al., 1999; Morris et al., 1999). The gene has three
exons and two introns. The ORF is 1602 bp long encoding a 57 kD enzyme.
Western analyses detected high levels of the enzyme in immature maize kernels.
Homologs of Arabidopsis were identified by BLAST searches. These homologs are
39% to 47% identical to the maize gene at the amino acid level and most of the
recombinant proteins have cytokinin oxidase activity (BiIyeu et al., 2001).
11
The enzyme mediating the formation of lupinic acid, the alanyl conjugate of
zeatin, has been partially purified from lupin seed (Entsch et at., 1980). The donor
substrate for the reaction is O-acetyl-L-serine. The Km for zeatin is 0.88 mM, one
or two orders of magnitude higher than the KmS of the other cytokinin enzymes.
The mass of the enzyme is about 64.5 kD.
A cis-trans isomerase of zeatin was partially purified from immature seed of
P. vulgaris. (Bassil et al., 1993). This enzyme favors conversion from the cis- to
the trans- isomer and therefore, the cis-trans isomerase has a possible role in
converting cis-zeatin to the more active trans-isomer (Mok and Mok, 2001). No
corresponding gene has been reported yet.
Reduction of trans-zeatin to dihydrozeatin is mediated by a zeatin
reductase. This enzyme was first isolated from immature seeds of P. vulgans
(Martin et al., 1989). The enzyme has high substrate specificity for trans-zeatin but
dose not appear to reduce cis-zeatin, frans-zeatin riboside, i6Ade, or zeatin 0glycosides. There are two isoforms of the enzyme, of about 25 kD and 55 kD,
respectively. Reduction of the side chain may preserve cytokinin activity in tissues
with high levels of cytokinin oxidase since dihydrozeatin is resistant to this
enzyme.
12
Objectives of this study
The general goal of this study was to contribute to our understanding of the
regulation of cytokinin 0-glucoside formation. The specific objectives were to
1) characterize the enzymatic reaction of cis-zeatin 0 glucosyltransferases and
2) explore the regulation of cytokinin 0-glycosyltransferase activity through
promoter-reporter gene fusions.
Following the isolation of cisZOGl encoding a cis-zeatin 0glucosyltransferase from maize, a second cis-zeatin 0-glucosyltransferase
(cisZOG2) was obtained from this species (R.C. Martin, M.C. Mok and D.W.S.
Mok, unpublished results). In this study, the biochemical characteristics of
cisZOGl and cisZOG2 were determined including the pH optimum for the reaction
and KmS for cis-zeatin and UDP-GIc. In addition, competition experiments were
performed to examine affinity to or inhibition by other cytokinins including tnszeatin, kinetin, BAP, i6Ade, cis-zeatin riboside, and thidiazuron.
To study the regulation of cytokinin 0-glycosyltransferase, the ZOX1 gene
was chosen since a genomic clone containing this gene has been isolated and the
region upstream of the ORE was sequenced (R.C. Martin, X. Shen, M.C. Mok and
D.W.S. Mok, unpublished results). This upstream segment (1.2 kb) was fused to
the f3-glucuronidase (GUS) gene (Jefferson, 1987). In this study, the construct was
inserted into the tobacco and Arabidopsis genome by Agrobacterium-mediated
transformation. GUS expression mediated by ZOX1 promoter in tissues of
transformed tobacco and Arabidopsis was determined, complementing previous
research on expression of ZOGI in Phaseolus (Martin et al., 1999a). The
13
availability of such transformants will facilitate future studies to analyse the effects
of cytokinins, other hormones, and environmental factors on the transcriptional
regulation of the ZOX1 promoter. Results of preliminary tests are presented.
14
References
Armstrong DJ (1994) Cytokinin oxidase and the regulation of cytokinin
degradation. In: Mok DWS and Mok MC (eds.) Cytokinins- chemistry, activity, and
function, pp. 139-154, CRC Press, Boca Raton.
Astot C, Dolezal K, Nordstrom, Wang Q, and Kunkel T (2000) An alternative
cytokinin biosynthesis pathway, Proc Nati Aced Sci USA 97: 14778-14783.
Barry GE, Rogers SG, Fraley RT, and Brand L (1984) Identification of a cloned
cytokinin biosynthetic gene, Proc NatI Aced Sd USA 81: 4776-4780.
Bassil NV, Mok DWS, and Mok MC (1993) Partial purification of a cis-trans
isomerase of zeatin from immature seed of Phaselous Vulgaris L., Plant Physiol
102: 867-872.
Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ et at. (2001) Molecular
and biochemical tharacterizaion of Maize, Plant Physiol, 125: 378-386.
Brzobohaty B, Moore I, Knstofferson P, Bakó L, Campos N et al. (1993) Release
of active cytokinin by a fl-glucosidase localized to the maize root meristem,
Science 262: 1051-1054.
Buchmann I, Mamer FJ, SchrOder G, Waffenschmidt S, and Schröder J (1985)
Tumor genes in plants: 1-DNA encoded cytokinin biosynthesis, EMBO J 4: 853859.
Campos N, Bakô L, Feldwisch J, Schell J, and Palme K (1992) A protein from
maize labelled with azido-IAA has novel /3-glucosidase activity, Plant J 2:675-684.
Davies PJ (eds.)(1995) Plant hormones; physiology, biochemistry, and molecular
biology, 2nd edition, Kluwer Academic Publishers, Dordredht.
Dixon SC, Martin RC, Mok MC, Shaw G, and Mok DWS (1989) Zeatin
glycosylation enzymes in Phaselous: isolation of 0-glucosyltransferase from P.
lunatus and comparison to O-xylosyltransferase from P. vulgaris, Plant Physiol 90:
1316-1321.
Einset JW (1984) Conversion of N6-isopentenyladenine to zeatin by Act inidia
tissues, Biochem Biophys Res Commun 124: 470-474.
Entsch B, Parker CW, Letham DS, and Summons RE (1979) Preparation and
characterization using high-performance liquid chromatography of an enzyme
forming glucosides of cytokinins, Biochem Biophys Acta 570: 124-139.
15
Entsch B and Letham DS (1979) Enzymic glycosylation of the cytokinin, 6-benzylaminopurine, Plant Sci Left 14: 205-2 12.
Entsch B, Letham DS, Parker CW, Summons RE, and Goilnow BI (1980)
Metabolites of cytokinins, In: Skoog F (eds.)(1979) Plant growth substances,
Biochem Biophys Acta 570: 124-1 39.
Gan S and Amasino RM (1996) Inhibition of leaf senescence by autoregulated
Production of Cytokinin, Science 270(22): 1986-1988.
Gersani M and Kende H (1982) Studies on cytokinin-stimulated translocation in
isolated bean leaves, Plant Growth Regul 1:161-171.
Gray J, Wang J, and Gelvin SB (1992) Mutation of the miaA gene of
Agrobacterium tumefaciens results in reduced vir gene expression, J. Bactenol
174: 1086-1098.
Guivarc'h A, Rembur J, Goetz M, Roitsch T, Noin M, Schmulling 1, and Chriqui D
(2002) Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum
L. cv. SRi) axillary buds establishes a role for cytokinins in tubenzation and sink
formation, J Experimental Botany 53(369): 621-629.
Houba-Henn n, Pethe C, dAlayer J, Laloue M (1999) Cytokinin oxidase from Zea
mays: puriifcation, cDNA cloning and expression in moss protoplasts, Plant J 17:
615-626.
Jefferson RA, Kavanagh TA, and Bevan MW (1987) GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants,
EMBO J 6: 3901-3907.
Kaiss-Chapman RW and Moms RO (1977) T,ans-zeatin in culture filtrates of
Agrobacterium tumefacien, Biochem Biophys Res Comm 76: 453-459.
Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as
Dimethylallyl Diphosphate: ATP/ADP isopentenyltransferases, Plant Cell Physiol
42(7): 1-111.
Letham DS, Wilson MM, Parker CW, Jenkins ID, MacLeod JK, and Summons RE
(1975) Regulators of cell division in plant tissues. XXII. The identity of an unusual
metabolite of 6-benzylaminopurine, Biochem Biophys Acta 399: 61-70.
Letham DS, Palni LMS, Tao GQ, Golinow BI, and Bates CM (1983) Regulators of
cell division in plant tissues. XXIX. The activities of cytokinin glucosides and
alanine conjugates in cytokinin bioassay, Plant Growth Regul 2:103-115.
16
Letham YY (1988) Plant senescence processes and free radicals, Free Radical
Biology and Medicine 5: 39-49.
Letham DS and Zhang R (1989) cytokinin translocation and metabolism in lupin
species. II. New nucleotide metabolites of cytokinins, Plant Sci 64: 161-165.
Letham DS (1994) cytokinins as phytohormones-sites of biosynthesis,
translocation, and function of translocated cytokinin, In: Mok DWS and Mok MC
(eds.) (1994) Cytokinins- chemistry, activity, and Function, pp. 57-80, CRC Press,
Boca Raton.
Martin RC, Mok MC, Shaw G, and Mok DWS (1989) An enzyme mediating the
conversion of zeatin to dihydrozeatin in Phaselous embryos, Plant Physiol 90:
1630-1635.
Martin RC, Mok MC, and Mok DWS (1999a) Isolation of cytokinin gene, ZOG1,
encoding zeatin 0-glucosyltransferase of Phaselous Iunatus, Proc Nati Aced Sd
USA 96: 284-289.
Martin RC, Mok MC, and Mok DWS (1999b) A gene encoding the cytokinin
Enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris, Plant Physiol 120: 553558.
Martin RC, Mok MC, Habben JE, and Mok DWS (2001) A maize cytokinin gene
encoding an 0-glucosyltransferase specific to cis-zeatin, Proc Nati Aced Sci USA
98(10): 5922-5926.
Miller CO, Skoog F, von Saltza MH, and strong M (1955) Kinetin, a cell division
factor from deoxyribonucleic acid, J Am Chm Soc 77: 1329-1334.
Mok MC and Mok DWS (1987) metabolism of 14C-zeatin in Phaselous embryos.
Occurrence of O-xylosyldihydrozeatin and its ribonudeoside, Plant Physiol 84:
596-599.
Mok DWS and Mok MC (eds.)(1 994) Cytokinins- chemistry, activity, and function,
CRC press, Boca Raton.
Mok MC and Mok DWS (2001) CYTOKININ METABOLISM AND ACTION, Annu
Rev Plant Physiol Plant Mol Biol 52: 89-118.
Moms RO, Bilyeu KD, Laskey JG, and Cheikh NN (1999) Isolation of a gene
encodong a glycosylated cytokinin oxidase from maize. Biochem Biophys Res
Commun 255: 328-333.
Nielsen TH and Ulvskov P (1992) Cytokinins and leaf development in sweet
pepper (Capsicum annuum L.) II. Sink metabolism in relation to cytokininpromoted leaf expansion, Planta 188: 78-84.
17
Paces V, Werstiuk E, and Hall RH (1971) conversion of N6-(2-isopentenyl)
adenosine to adenosine by enzyme activity in tobacco tissue, Plant Physiol 48:
775-778.
Richmond A and Lang A (1957) Effect of kinetin on protein content and survival of
detached Xanthium leaves, Science 125: 650-651.
Schmulling T, Beinsberger S, De Greef J, Schell J, Van Onckelen H, and Spena A
(1989) Construction of a heat inducible chimaenc gene to increase the cytokinin
content in transgenic plant tissue, FEBS Lett 249: 401-406.
Skoog F and Miller CO (1957) Chemical regulation of growth and organ formation
in plant tissues cultured in vitro, Symp Soc ExpI Biol 11: 118-131.
Skoog F and Armstrong DJ (1970) Cytokinins, Annu Rev Plant Physiol 21: 359384.
Smigocki AC and Owens LD (1988) Cytokinin gene fused with a strong promoter
enhances shoot organogenesis and zeatin levels in transformed plant cell, Proc
Nati Aced Sci USA 85: 51 31-51 35.
Stmad M (1997) The aromatic cytokinins. Plant Physiol 101: 674-688.
Takei K, Takahashi 1, Sugiyama T, Yamaya T, and Sakakibara H (2002) Multiple
routes communicating nitrogen availability from roots to shoots: a signal
transduction pathway mediated by cytokinin, J Experimental Botany 53 (370): 971977.
Tantikanjana T, Yong JWH, Letham DS, Griffith M, Hussain M, Ljubg K, Sandberg
G, and Sundaresan V (2001) Control of axillary bud initiation and shoot
architecture in Arabidopsis through the SUPERSHOOT gene, Genes and
Development 15(12): 1577-1588.
Taya Y, Tanak Y, and Nishimura S (1978) 5'-AMP is a direct precursor of cytokinin
in Dictyostelium discoidum, Nature 271(541): 545-547.
Thimann Ky (1987) Plant senescence: A proposed integration of the constituent
processes, In: Thomson W\N, Nothnagel EA, and Huffaker (eds.) Plant
senescence: Its biochemistry and Physiology, pp. 1-19, American society of plant
physiologists, Rockville, Md.
Thomson JE, Legge RL, and Barber TF (1987) The role of free radicals in
senescence and wounding, New Physiologist 105: 317-344.
18
Turner JE, Mok DWS, Mok MC, and Shaw G (1987) Isolation and partial
purification of an enzyme catalyzing the formation of O-xylosylzeatin in Phase/ous
vulgaris embryos, Proc NatI Acad Sci USA 84: 3714-3717.
Van Staden J, Cook E, and Noodén LD (1988) Cytokinins and senescence, In:
Noodén LD and Leopold AC (eds.) Senescence and aging in plants, pp. 281-328,
Academic Press, San Diego.
Vañková R (1999) Cytokinin glycoconjugates-distribution, metabolism and
function, In: Strand M, Pee, Beck E (eds.) Advances in Regulation of Plant Growth
and Development, pp. 67-78, Prague, Peres.
Werner T, Motyka V, Stmad M, and Schmulling T (2001) Regulation of plant
growth by cytokinin, Proc NatI Aced Sd USA 98(18): 10487-10492.
Zubko E, Adams C, Macháèková I, Malbeck J, Scollan C, and Meyer P (2002)
Activation tagging identifies a gene from Petunia hybrida responsible for the
production of active cytokinins in plants, Plant J 29(6): 797-808.
19
CHAPTER 2
BIOCHEMICAL CHARACTERIZATION OF
cis-ZEATIN 0-GLUCOSYLTRANSFERASES, cisZOGl AND cisZOG2
Abstract
tns-Zeatin is the major and ubiquitous cytokinin in higher plants. cisZeatin has traditionally been viewed as an adjunct with low activity and rare
occurrence. Recent reports of cis-zeatin and derivatives as the predominant
cytokinin components in some plant tissues necessitate a different perspective on
the role of cis-isomers. The cloning of a maize gene (cisZOGl) encoding an 0glucosyltransferase specific to cis-zeatin (Martin et al., 2001) lends support to this
view. This chapter deals with charactenzation of biochemical parameters of
cisZOGl as well as c1sZOG2, a recently isolated homolog of cisZOGl. cisZOG2
has 98% identity to cisZOGl and the upstream regions contain common and
unique segments. The recombinant enzymes have similar properties, pH optimum
of 7.5 and Kms of 46 and 96j.tM respectively for cis-zeatin. N6(A2
isopentenyl)adenine, cis-zeatin riboside, and thidiazuron were strong inhibitors of
the reaction while the presence of tns-zeatin, benzyladenine, and kinetin had
only slightly inhibitory effect. The results, together with the recent identification of
cis-isomers in maize (R. Vankova, J. Malbeck, R.C. Martin, D.W.S. Mok and M.C.
Mok, unpublished results) are a clear indication that cis-zeatin O-glucosylation is a
20
natural metabolic process in maize. Whether cis-zeatin serves as a precursor to
the active trans-isomer or has any other unique function remains to be
demonstrated.
Introduction
Cytokinins are plant hormones regulating cell division and a host of
developmental events such as bud formation, leaf expansion, senescence, seed
germination, and chloroplast formation (Mok, 1994). trans -Zeatin is the major and
ubiquitous cytokinin in higher plants. Earlier cytokinin analyses detected cis-zeatin
and its derivatives in trace amounts in some plants but due to their low activity
(Schmitz et al., 1972), cis-isomers were viewed as adjunct to trans-isomers.
Recent analyses, however, showed that the cis-isomers can be the dominant
cytokinins at particular stages of developmept in plants such as chickpea and
lupine (Emery et al., 1998; Emery et at., 2000). Moreover, the presence of cisisomers was associated with male stenlity in Mercunalis flowers (Louis et at., 1990;
Durand and Durand, 1994). These are indications that cis-isomers may have
unique physiological functions, although as yet unknown. The need to regulate the
levels of cis-zeatin is evidenced by the maize cisZOGl gene, encoding an 0glucosyltransferase with specificity to cis-zeatin (Martin et al., 2001).
0-Glucosylation is a major step in the metabolism of trans-zeatin (Mok and
Mok, 2001). The resulting 0-glucosides serve as reversible storage compounds
and are resistant to degradation by cytokinin oxidases (Armstrong, 1994). These
21
0-glucosides are found in xylem sap and are also likely transport forms of zeatin
(Letham, 1994). Phaseolus enzymes and genes involved in conversion of
trans -
zeatin to its 0-glucoside and O-xyloside have been characterized in our
laboratories (Turner et aL, 1987; Dixon et al., 1989; Martin et al., 1999a,b). These
dicot enzymes have no or very low affinity to cis-zeatin. Therefore, the discovery of
the maize cisZOGl gene is indicative of the possible existence of parallel
metabolic pathways between tmns- and cis-zeatin, serviced by isomer-specific
enzymes. Recently, a second cisZOG gene was isolated from maize (R.C. Martin,
M.C. Mok and D.W.S. Mok, unpublished results). Here the biochemical
characteristics of cisZOGl and cisZOG2 are reported, including the pH optimum,
Kms for cis-zeatin and UDP-Glc, and inhibitory effects of other cytokinins on the
conversion of cis-zeatin by this type of enzyme.
Materials and Methods
Generation of recombinant protein
The ORF of cisZOG2 was amplified by PCR with a forward primer specific
to this gene containing an Ncol site (GAG CTC CAT GGC GGT TGA CAC GAT
GGA) and a backward primer containing an Xbal site (GAA TTC ACT CTA GAT
TAC CU GTG ATG TAG CCA ATG). The PCR product was digested with Ncol
and XbaI and ligated into the Ptrc99A vector (Amersham Pharmacia), which was
22
modified to contain a translational fusion with seven histidine residues at the N
terminus of the protein. This vector was transformed into the XLI Blue cell line.
The same procedures as used for generating crude cisZOGl enzyme (Martin et
al., 2001) were used to obtain cisZOG2 protein. Both cisZOGl and cisZOG2
enzymes were purified by incubation with Ni column material (Novagen) for 2 hr at
4°C, followed by sequential elution with 100mM and 500mM imidazole. The 500
mM imidazole fraction contained the purified enzyme, which was concentrated by
ultrafiltration in Centnprep 10 (Millipore) and desalted on an Econo-Pac 10 DG
column (BioRad).
Biochemical characterization of recombinant enzymes
Enzyme activity was assayed by incubating 0.75p.M cis-[3H]zeatin (specific
activity 3.lCi/mmol), 4mM UDP-Glc, lOpi retombinant protein, and 0.17M MgCl2in
l5OpJ 0.17M Tris, pH 7.5, for 30 mm or 1 hr at 27°C. The reaction was terminated
by addition of 800
jtl
cold 95% ethanol, after which the mixture was centrifuged and
the supernatant collected. Substrate and product were separated by HPLC (Dixon
et al., 1987). For determination of the optimum pH for the reaction, the pH range of
6.5-8.5 was used. For Km determinations of cis-zeatin various amounts of nonradiolabeled cis-zeatin were added and for Km determination of UDP-Glc the cis-
zeatin concentration was 100iM. The reaction was linear over time (30 mm) at the
chosen concentrations. Km values were determined with Scientist Micromath
version 2.0 from Micromath Inc. To determine possible inhibition by other
23
cytokinins, I OOtM of the compound was added to the reaction mixture containing
O.75tM cis-[3H]zeatin.
Results
Description of cisZOGl and cisZOG2
The cisZOGl and cisZOG2 genes are 1404 and 1392 bp long and have
98.3% identity. Neither cisZOGl nor cisZOG2 have any introns. The deduced
amino acid sequence of cisZOG2 is 98% identical to that of cisZOGl, but the
protein is shorter by four amino acids (Fig. 2.1).
c is ZOG1 1 MAVDTMESVAVVAVPFPAQGHLNQLLHLSLLLASRGLSVHYAAPPPHVRQ
50
111111111111111111111 III III I
I 111111
1111
cisZOG2 1 MAVDTMESVAVVAVPFPAQGHLNQLLHLSLLLASRGLSVHYAAPPPHVRQ
50
I
I
I
I
I
I
51 ARARVHGWDPRALGSIRFHDLDVPPYDSPAPDL7.APSPFPNHLMpMFEAF 100
II
1111111111111 II till ii 11111111111
1111111
51 ARARVHGWDPRALGS I}IFHDLDVPAYDSPAPDLAAPSPFPMHLMPMFEAF 100
I
101 AAAARAPLAALLQRLSTSYRRVAVVFDRLNPFATEAARLANADAFGLQC 150
Ill
Ill liii 11111111
111111111
101 AAAARAPLAALLQRLSTSYRRVAVVFDRLNPFATEAARLANADAFGLQC 150
151 VAISYNVGWLDPGHRLLSDYGLQFLPPDACMSREFVDLVFRMEEEEQGAP 200
11111
III
11111111111 I
I
I
I
11111111111 I
11111
I
151 VAISYTVGWLDPGHRLLSDYGLQFLPPDDCMSREFVDLVFRMEEEEQGAP 200
201 VAGLVMNTCRALEGEFLDVVAAQPPFQGQRFFAVGPLNPLLLDADAPTTP 250
201 VAGLVMNTCRALEGEFLDAVAAQPPFQGQRFFAVGPLNPLLLDADARTAP 250
251 PGQARHECLEWLDRQPPESVLYVSFGTTSCLHADQVAELAAALKGSKQpy 300
251
111111
11111 1111111! lii 11111111111 I 11111111
. . . . RHECLEWLDRQPPESVLYVSFGTTSCLHADQVAELAAALIcGSjcQRF 296
24
301 VWVLRDADRADIYAESGESRHAMFLSEFTRETEGTGLVITGWAPQLEILA 350
:1
1111111111 II 1:11111111 II
11111111111111
297 VWVLRDADRADIYAESGDSRHAKFLSEE1RETEGTGLVVTGWAPQLEILA 346
I
I
351 HGATPAE}1SHCGWNSTIESLSHGKPVLAWPMHSDQPWDSELLCKYEKGL 400
11111111111 III 1111111111111111111111 liii 111111
347 HGATAE'MSHCGWNSIIESLSHGKPVLAWPMHSDQPWDSELLCNYFKGL 396
401 LVRPWEKHAEIVP1QAIQKVIEEAMLSDSGMAVRQRAKELGEAVRASVAD 450
1111111111
11111111111: 11111 11111111111 III liii
397 LVRPWEKHAEIIPAQAIQKVIEEAMLSDSGMAVRQRAKELGEAVRASVAD 446
451 GGNSRKDLDDFIGYITR* 467
liii
111111
liii
447 GGNSRKDLDDFIGYITR* 463
Figure 2.1. Comparison of the sequences of cisZOGl and cisZOG2 genes.
Alignment of the deduced amino acid sequences.
However, the upstream regions of the two genes are different, with large
gaps in cisZOG2, although there are also highly similar regions (Fig. 2.2). Both
recombinant enzymes have a theoretical p1 of 5.4 and mass of 51 kD.
-1214
-1128
-90 -72
-29 -6
cIsZOGl
94.1%
84.2%
cIsZOG2--........
-131
-47
-45 .27
95.8%
-
-26 -3
Figure 2.2. Diagrammatic presentation of the upstream sequences.
Alignment was performed with the Genetics Computer Group software
(Madison, WI).
The open reading frames (OREs) of both genes were cloned into the
modified expression vector Ptrc99A producing recombinant proteins with a
histidine tag at the N-terminus. Incubation of cis-zeatin with crude enzyme resulted
in formation of cis-zeatin 0-glucoside but additional products were formed (Fig.
2.3). When enzyme was purified over a Ni column, only the 0-glucoside was
obtained (Fig. 2.3). This purified extract was used to study the biochemical
properties of the enzyme. The recombinant proteins catalyze the formation of 0glucosyl cis-zeatin with a pH optimum of 7.5 (Fig. 2.4).
26
20000
30000
-
A
15000
20000
E
10000
0.
0
10000
5000
0
35
40
45
50
55
40
35
60
50
45
55
60
mmn
mm
100000
40000
C
80000
30000
E 20000
0
J
0-glucosyl
\ cis-[H1zeatin
E
D
0-glucosyt
cis-[3HJZeatin
60000
I
cis-[3HlZeatin
40000
\
I
7\
cis-[3H]Zeatin
20000
$
mm
miii
Figure 2.3. Separation by HPLC of the substrate (cis-[H3Jzeatin) and product
(0-glucosyl cis-[H3jzeatin) obtained from the cisZOG enzyme reaction.
A. Crude extract of cisZOGl. B. Crude extract of cisZOG2. C. Ni column
purified extract of cisZOGl. D. Ni column purified extract of cisZOG2.
C
0
70
60
C
8
50
6
6.5
7
7.5
8
8.5
9
pH
B
6 67758&5 9
Figure 2.4. The effect of the pH on the conversion of c/s-[3H]zeatin to
cis-[3H]zeatin 0-glucoside by cisZOGl(A) and cisZOG2(B).
27
The Km for cis-zeatin is about 46xM for cisZOGl and 96p.M for cisZOG2.
UDP-glucose (UDP-Glc) is the sugar donor with Kms of 0.11 and 0.59mM for
cisZOGl and cisZOG2, respectively (Fig. 2.5; Table 2.1).
B
15
10
8
10
6
8
4
>
2
100
0
200
300
400
L.oncenlrauon (UM)
Concentration (tiM)
50
25
20
IC
40
D
30
15
20
8 10
10
S
oncenirrnuun (UM)
Loncenlratuon
(tiM)
Figure 2.5. The effect of substrate concentration on the velocity of
glucosyl transfer by cisZOGl and cisZOG2. A. cis-zeatin as substrate,
with 4mM UDP-Glc and 13.5p.g cisZOGl protein. B. cis-zeatin as
substrate, with 4mM UDP-GIc and 8.2p.g cisZOG2 protein. C. UDP-Glc
as substrate, with lOOp.M cis-zeatin and 27p.g cisZOGl protein. D. UDPGic as substrate, with lOOp.M cis-zeatin and 98.8ig cisZOG2 protein.
Samples were incubated for 30 mm at 27°C and pH 7.5.
28
Table 2.1. Km and Vmax values of cisZOGl and cisZOG2 for
cis-zeatin and UDP-Glc. Calculations were preformed using
Scientist Micromath version 2.0 from Micromath Inc.
Km
cisZOG I
cisZOG2
Vmax
cis -Zeatin
46tM
54tkat/kg
UDP-GIc
11OiM
31ikat/kg
96p.M
129tkatJkg
593j.tM
40j.tkat/kg
CIS
-Zeatin
UDP-Glc
UDP-xylose (UDP-Xyl) does not serve as the sugar donor, another
property distinguishing these enzymes from trans-zeatin 0-glucosyltransferases of
Phaseolus,
which can transfer the xylose from UDP-Xyl albeit at a lower affinity
than UDP-GIc (Dixon et al. 1989, Martin et al. 1 999ab). Using larger quantity of
purified enzyme, formation of trans-[3H]zeatin 0-glucoside was mediated by both
maize enzymes under standard reaction conçiitions. However, conversion of cis-
zeatin was I 1-fold higher than that of trans-zeatin under the same conditions. In
addition, cis-zeatin nboside could be converted to the corresponding 0-glucoside
when purified recombinant enzyme was used.
To examine possible affinity to or interference by other cytokinins,
competition experiments were conducted. Conversion of cis-zeatin to its glucoside
was slightly lowered by the presence of trans-zeatin, kinetin and benzyladenine,
while isopentenyladenine and cis-zeatin riboside caused significant reduction (Fig.
2.6). Interestingly, thidiazuron, a very active phenylurea-type cytokinin, also
decreased conversion of cis-[3H]zeatin. Thidiazuron also inhibits cytokinin oxidase
29
activity (Armstrong, 1994) and was shown to be a competitive inhibitor of the
maize oxidase (Bilyeu et al., 2001). Whether the inhibition of glucosyltransferase
activity observed here is competitive or non-competitive remains to be determined
but it appears that thidiazuron interferes with a host of cytokinin-specific but
distinct enzymatic reactions.
100
0
C
0
0
0
80
60
40
C
0
U)
C
0
20
0
0
C
cZ cZR
tZ iAde BAP K
Ade TDZ
Figure 2.6. Cytokinins as competitors of the conversion of cis-[3H]zeatin
to its glucoside by cisZOGl. Enzyme activity was assayed by incubating
0.75tM cis-[3H]zeatin (specific activity 3.1 Ci/mmol), I OOp.M cytokinin,
4mM UDP-Glc, lOpi recombinant protein, and 0.17M MgCl2in 150p1
0.17M Iris, pH 7.5, for 30 mm or 1 hr at 27°C. Abbreviations: C, control
(no cytokinin added); cZ, cis-zeatin; cZR, cis-zeatin riboside;
tZ, trans-zeatin; i6Ade, N6-(A2-isopentenyl)adenine; BAP, N6benzyladenine; K, kinetin; Ade, adenine; TDZ, thidiazuron.
30
Discussion
The clear preference of the two glucosyltransferases for cis-zeatin
indicates that cis-zeatin 0-glucosylation is regulated by specific enzymes. The KmS
for cis-zeatin and UDP-Glc are well within the range expected of cytokinin
metabolic enzymes. For example, the recombinant cytokinin oxidase of maize has
KmS of 46tM for cis-zeatin and 14tM for trans-zeatin (Bilyeu et al., 2001), while
other, native cytokinin oxidases have reported Km values for trans-zeatin ranging
from 25 to 33p.M (Armstrong, 1994). The Km of the native zeatin 0
glucosyltransferase of P. lunatus is 28jtM for trans-zeatin and 0.2mM for UDP-Glc
(Dixon et al., 1989). The difference in Km between cisZOGl and cisZOG2 may be
related to the gap of four amino acids in cisZOG2, resulting in slight differences in
folding, or in the additional amino acid differences. With larger amounts of
recombinant enzyme available, cis-zeatin riboside was also found to be a
substrate. However, the K, was not determined for lack of radiolabeled cis-zeatin
riboside.
The cytokinin 0-glycosyltransferases identified thus far have high substrate
specificity, differentiating between trans- and cis-zeatin, and between sugar
donors, UDP-Glc or UDP-Xyl (Dixon et al., 1989; Martin et al., 1999a,b). The
genes encoding these enzymes have higher homology to each other than to any
other sequences in the GenBank. Among glycosyltransferase genes, the cytokinin
0-glycosyltransferases constitute a distinct branch in the evolutionary tree (Li et
al., 2001). The upstream regions of the cisZOGl and cisZOG2 have stretches with
31
high identity but also unique segments. It is not known as yet if these differences
signify differential gene expression.
Parallel to this study, the endogenous cytokinins in maize tissues were
analyzed (Vankova R, Malbeck J, Martin RC, Mok MC, Mok DWS, unpublished
results). The analyses indicated that cis-zeatin was clearly present in roots, stems,
leaves, unfertilized cobs, and kernels along with its riboside and nucleotide. The
0-glucosides of cis-isomers were found in roots, young cobs, and kernels,
compatible with the earlier observation that cisZOGl expression is also high in
these tissues (Martin et al., 2001). It was somewhat surprising to find high levels of
cis-zeatin derivatives in maize tissues since maize has been the subject of
numerous cytokinin analyses in the past and the occurrence of cis-zeatin has, to
my knowledge, never been reported. However, in rice, another monocot, 0glucosyl cis-zeatin and its riboside were found (Takagi et al., 1989). In fact, the
number of species in which cis-zeatin was detected has increased steadily over
the years and now includes potato (Mauk and Langille, 1978; Suttle et al., 2000),
hops (Watanabe et al., 1981), rice (Takagi et al., 1985), wheat (Parker et al.,
1989), oats (Parker et al., 1989), Mercurialis (Durand and Durand, 1994), chickpea
(Emery et al., 1998), lupins (Emery et al., 2000), and tobacco (Dobrev et al.,
2002). Although many other studies did not report the presence of cis-isomers,
that is more likely due to their exclusion from cytokinin analyses rather than their
absence from the tissues. It is reasonable to assume that they are present in all
higher plants, but probably vary in abundance.
The possible origin of high level of cis-isomers is not known. Hydrolysis of
cytokinin-containing tRNAs (Skoog and Armstrong, 1970) is a possibility but is
32
probably insufficient to account for the amount detected recently (for more
detailed discussion, see Mok and Mok, 2001). Another is the direct synthesis via a
c/s-hydroxylated precursor side chain, similar to the proposed hydroxylated side
chain leading to the synthesis of trans-zeatin (Astot et al., 2000; Mok and Mok,
2001). Although naturally occurring derivatives of dimethylallyl pyrophosphate
hydroxylated in either the trans- or cis-configuration have not been reported, the
deoxyxylulose phosphate pathway of isoprenoid biosynthesis, (Lichtenthaler,
1999) has as intermediates compounds that can potentially yield both cis- and
trans-hydroxylated side chains. Isopentenyltransferases (Kakimoto, 2001; Takei et
al., 2001) may mediate the direct synthesis of cis- and trans-zeatin riboside
phosphate from such precursors. It is also possible that substrate specificity
governs such reactions, similar to 0-glucosylation, with separate enzymes
mediating the formation of cis- and trans-zeatin.
The presence of high levels of cis-isomers and their regulation by specific
enzymes are puzzling considering that cis-zeatin is only weakly active in standard
cytokinin bioassays and no clear function has been assigned to cis-zeatin. It is
possible that at specific stages of development cis-zeatin is converted to the more
active isomer by a cis-trans zeatin isomerase (Bassil et al., 1993). In this context,
early abundance of cis-isomers in developing chickpea seed followed by an
increase in trans-isomers (Emory et al., 1998) can be viewed as cis-zeatin
contributing to the highly active cytokinin pool. However, direct conversion from
cis- to trans-isomers has yet to be demonstrated. cis-Zeatin could possibly also be
converted to dihydrozeatin, by a reductase similar to the trans-zeatin reductase of
Phaseolus (Martin et al., 1989). Alternatively, cis-zeatin may have some
33
specialized function as yet unidentified. For instance, the presence of cis-zeatin is
associated with male sterility in
Mercurialis (Louis
et al., 1990; Durand and
Durand, 1994). Finally, cis-zeatin could potentially down-regulate cytokinin activity
by acting as a competitor to trns-zeatin through binding to specific enzymes or
receptors. In any event, the abundance of cis-zeatin and cis-specific enzymes
indicates the need to re-examine the role of cis-zeatin and derivatives in cytokinin
biology.
34
References
Armstrong DJ (1994) Cytokinin oxidase and the regulation of cytokinin degradation,
In: Mok DWS and Mok MC (eds.) Cytokinins- chemistry, activity, and function,
pp.139-154, CRC Press, Boca Raton.
Astot C, Dolezal K, NordstrOm A, Wang Q, Kunkel T, Montz T, Chua NH, and
Sandberg C (2000) An alternative cytokinin biosynthesis pathway, Proc Nati Aced
Sd USA 97:14778-14783.
Bassil NV Mok DWS, and Mok MC (1993) Partial purification of a cis-trans
isomerase of zeatin from immature seed of Phaseolus vulgaris L., Plant Physiol
102: 867-872.
Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ, Kramer MD, and Morris
RO (2000) Molecular and biochemical characterization of a cytokinin oxidase from
Zea mays, Plant Physiol 125: 378-386.
Dixon SC, Martin RC, Mok MC, Shaw C, and Mok DWS (1989) Zeatin
glycosylation enzymes in Phaseolus: isolation of 0-glucosyltransferase from P.
lunatus and comparison to O-xylosyltransferase from P. vulgaris, Plant Physiol
90: 1316-1321.
Dobrev P, Motyka V, Gaudinová A, Malbeck J, Trávniková A, Kaminek M, and
Vanková R (2002) Transient accumulation of cis- and trans-zeatin type cytokinins
and its relations to cytokinin oxidase activity during cell cycle of synchronized
tobacco cells, Plant Physiol Biothem 40: 333-337.
Durand R and Durand B (1994) Cytokinins ard reproductive organogenesis in
Mercunalis, frj Mok DWS and Mok MC (eds.) Cytokinins; chemistry, activity, and
function, pp. 295-304, CRC Press, Boca Raton.
Emery RJN, Leport L, Barton JE, Turner NC, and Atkins CA (1998) cis-lsomers of
cytokinins predominate in chickpea seeds throughout their development, Plant
Physiol 117: 1515-1523.
Emery RJN, Ma Q, and Atkins CA (2000) The forms and sources of cytokinins in
developing white lupine seeds and fruits, Plant Physiol 123:1593-1604.
Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as
dimethylallyl diphosphate:ATP/ADP isopentenyltransferases, Plant Cell Physiol
42: 677-685.
Letham DS (1994) Cytokinins as phytohormones sites of biosynthesis,
translocation, and function of translocated cytokinin, In: Mok DWS and Mok MC
(eds.) Cytokinins; chemistry, activity, and function, pp. 57-80, CRC Press, Boca
35
Raton.
Li Y, Baldauf S, Lim E-K, and Bowles DJ (2001) Phylogenetic analysis of the UDPglycosyltransferase multigene family of Arabidopsis thaliana, J Blot Chem
276: 4338-4343.
Lichtenthaler IlK (1999) The 1-deoxy-D-xytutose-5-phosphate pathway of
isoprenoid biosynthesis in plants, Annu Rev Plant Physiol Plant Mol Blot 50: 4765.
Louis J-P, Augur C, and Teller G (1990) Cytokinins and differentiation processes in
Mercurialis annua, Plant Physiol 94: 1535-1541.
Martin RC, Mok MC, Habben JE, and Mok DWS (2001) A cytokinin gene from
maize encoding an 0-glucosyltransferase specific to cis-zeatin, Proc NatI Acad
Sd USA 98: 5922-5926.
Martin RC, Mok MC, and Mok OWS (1999a) Isolation of a cytokinin gene, ZOGI,
encoding zeatin 0-glucosyltransferase of Phaseolus iunatus, Proc NatI Acad Sd.
USA 96: 284-289.
Martin RC, Mok MC, and Mok DWS (1999b) A gene encoding the cytokinin
enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris, Plant Physiol 120:
553-557.
Martin RC, Mok MC, Shaw G, and Mok DWS (1989). An enzyme mediating the
conversion of zeatin to dihydrozeatin in Phaseolus embryos, Plant Physiol
90: 1630-1635.
Mauk CS and Langille AR (1978) Physiology of tuberization in Solanum tube rosum
L. Cis-zeatin nboside in the potato plant: its identification and changes in
endogenous levels as influenced by temperature and photoperiod, Plant Physiol
62: 438-442.
Mok DWS and Mok MC (1987) Metabolism of 14C-zeatin in Phaseolus embryos,
Plant Physiol 84: 596-599.
Mok DWS and Mok MC (2001) Cytokinin metabolism and action, Annu Rev Plant
Physiol. Plant Mol Biol 52: 89-118.
Mok MC (1994) Cytokinins and plant development an overview, In: Mok DWS,
Mok MC (eds.) Cytokinins; chemistry, activity, and function, pp. 155-166, CRC
Press, Boca Raton.
Parker CW, Badenoch-Jones J, and Letham OS (1989) Radioimmunoassay for
quantifying the cytokinins cis-zeatin and cis-zeatin riboside and its application to
xylem sap samples, Plant Growth Regul 8: 93-105.
36
Schmitz RY, Skoog F, Playtis AJ, and Leonard NJ (1972) Cytokinins: synthesis
and biological activity of geometric and position isomers of zeatin, Plant Physiol
50: 702-705.
Skoog F and Armstrong DJ (1970) Cytokinins, Ann Rev Plant Physiol 21: 359384.
Suttle JC and Banowetz GM (2000) Changes in cis-zeatin and cis-zeatin riboside
levels and biological activity during tuber dormancy, Physiol Plant 109: 68-74.
Takagi M, Yokota T, Murofushi N, Ota Y, and Takahashi N (1985) Fluctuation of
endogenous cytokinin contents in rice during its life cycle - quantification of
cytokinins by selected ion monitoring using deuterium-labeled internal standards,
Agric Biol Chem 49: 3271-3277.
Takagi M, Yokota T, Murofushi N, Saka H, and Takahashi N (1989) Quantitative
changes of free-base, riboside, nbotide and glucoside cytokinins in developing rice
grains, Plant Growth Regul 8: 349-364.
Takei K, Sakakibara H, and Sugiyama T (2001) Identification of genes encoding
adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis
thaliana, J Biol Chem 276: 26405-26410.
Turner JE, Mok OWS, Mok MC, and Shaw G (1987) Isolation and partial
purification of an enzyme catalyzing the formation of O-xylosylzeatin in Phaseolus
vulgaris embryos, Proc Natl Acad Sd USA 84: 3714-3717.
Watanabe N, Yokota T, and Takahashi N (181) Variations in the levels of cis- and
tmns-nbosylzeatins and other minor cytokinins during development and growth of
cones of the hop plant, Plant Cell Physiol 22: 489-500.
37
CHAPTER 3
PROMOTER STUDY OF A GENE ENCODING
A trans-ZEATIN 0-GLYCOSYLTRANSFERASE
Abstract
Cytokinin 0-glucosides are assumed to be the reversible storage forms of
active cytokinins since they can be readily converted to the aglycones by
I-
glucosidases. A gene encoding a glucosyltransferase responsible for the formation
of 0-glucosylzeatin (ZOGI) was isolated from Phaseolus lunatus seed (Martin et
al., 1999a) and a zeatin O-xylosyltransferase gene, ZOX1, was cloned from P.
vulgavis (Martin et al., 1999b). In this study, transformed tobacco and Arabidopsis
were generated that contain the ZOX1 promoter fused to the GUS
glucuronidase) gene.
T1
(f-
progenies that wer PCR- and Southern-positive were
analyzed for GUS activity. In vegetative tissues of tobacco, pZOX1 mediated
reporter gene expression in trichomes and stem hairs, both originating from
epidermal cells. The endosperm of tobacco seeds containing the pZOX1::GUS
construct was also GUS-positive. Induction of promoter activity by cytokinin and
auxin was explored to a limited extent. It appears that GUS expression can be
activated by zeatin, benzyladenine, and naphthalenacetic acid (NAA) in trichomes
of leaves no longer expressing GUS. In contrast to tobacco, GUS activity was not
detected in pZOX1::GUS transformed Arabidopsis. These results indicated that
38
pZOX1-derived gene expression is species-specific and possibly restricted to
endomitotic cells such as endosperm and trichomes.
Introduction
Plant tissues contain an array of cytokinin metabolites and glucosides are
the most common derivatives. The glucosyl group can be attached to hydroxytated
side chain to form 0-glucosides, or to the adenine ring, giving rise to N-glucosides.
The 0-glucosides serve as reversible reserve since they can be readily converted
to the aglycone by f3-glucosidases. In contrast, N7- and N9-glucosylation represent
irreversible conjugation.
A glucosyltransferase responsible for the formation of 0-glucosyizeatin was
isolated from Phaseolus lunatus seed (Dixon et al., 1989) and a related enzyme,
zeatin O-xylosyttransferase, was cloned from P.
vulgaris
(Turner et at., 1987).
Western blot analyses and tissue prints using a monoclonal antibody specific to
these enzymes detected the presence of high levels of these glycosyltransferases
in immature seed, particularly in endosperm (Martin et at., 1993). Northern
analyses showed that steady-state mRNA levels are high in seeds, white levels
are lower in roots and very low in leaves (Martin et al., 1999a, b). The two
glycosylation genes have similar expression patterns. To determine more precisely
possible cell type-specific gene expression and to study gene induction by
chemical and environmental cues, promoter-reporter constructs in transgenic
plants were employed. This chapter describes the results of using the ZOX1
39
sequence containing the promoter fused to the GUS (-glucuronidase) gene to
generate transformed tobacco and Arabidopsis since transformation of Phaseolus
is extremely difficult. Preliminary results of promoter-mediated expression patterns
in these transgenic plants are presented.
Materials and Methods
Transformation vectors
The genomic sequence of ZOXI and a 1.2-kb fragment upstream from the
translation initiation site were obtained (Shan X, Martin RC, Mok MC, and Mok
DWS, unpublished results). The 1.2-kb fragment was fused to the J3
glucuronidase (GUS) gene in plasmid pBISNI (obtained from S.B. Gelvin, Purdue
University). A transient expression assay using particle bombardment
demonstrated that this fragment contains the promoter region since it mediated
GUS expression in immature bean seeds (Shan X, Martin RC, Mok MC, and Mok
DWS, unpublished results). The current study employed this pZOX1::GUS
construct as well as the pBISN1 plasmid containing the "superpromoter"
(SP::GUS) and the corresponding pasmid without promoter (noP::GUS). The
plasmids were moved into Agrobacterium tumefaciens (EHA1O5) according to the
methods of Hofgen and Willmitzer (1988). Kanamycin resistance was the
selectable marker.
40
Tobacco (W38) and Arabidopsis were used as the model systems as
transformation of Phaseo!us is still problematic.
Tobacco transformation
Nicotiana
tobacum L. cv. W38 was transformed with A. fume faciens
EHA1 05/pZOXI::GUS, EHAI O5ISP: : GUS, and EHA1 05/floP:: GUS. A.
fume faciens cultures were grown overnight in 8m1 YEP (with 1 00.tg m11
kanamycin) in a shaker/incubator at 250 rpm and 28°C. When the OD was 0.5,
the culture was diluted 1:10 in Murashige and Skoog (MS) medium without
hormones. The transformation procedure of Horsch et al (1988) was used. Bnefly,
leaf discs were incubated in the Agmbactenum-containing MS medium for 10 mm,
blotted dry on sterile paper towels to get rid of excess Agrobactrium on the
surface, and placed on regeneration medium (MS medium with 1mg L1 BAP and
0.1mg L1 NAA). After 48 hr incubation in a lighted culture room under a 16 hr /8 hr
(light / dark) cycle at 25°C, the leaf discs were transferred to fresh regeneration
medium with 500mg L1 kanamycin and 400mg
L1
timentin and incubated in the
lighted culture room. Shoots formed on leaf discs were excised and transferred to
rooting medium (MS medium with 500mg
L1
kanamycin and 400mg L1 timentin).
Plantlets were placed in the mist house for 2 weeks and then taken into the
greenhouse.
T1
seeds were placed on MS medium containing 500mg L1 kanamycin.
The numbers of resistant (green) and susceptible (white) seedlings were
41
determined. Green seedlings were transferred to soil and placed in the
greenhouse to determine GUS expression and to obtain T2 seeds.
Arabidopsis transformation
For transformation of Arabidopsis, the floral dip method (Bechtold et al.,
1993; Clough and Bent, 1998) was used. Agrobacterium was grOwn overnight in
8m1 LB (with 100.tg m11 kanamycin) at 28°C and 250 rpm. The next day, 2.5m1 of
this culture was diluted into lOOmI LB medium (with 100.ig m11 kanamycin) and
grown for 24 hr. After centrifugation at 6,000 rpm for 15 mm, the pellet was resuspended in 400m1 of a 5% sucrose solution with 8Op.l Silwet L-77. Arabidopsis
flowers were dipped in this Agrobacterium solution. After I week, the procedure
was repeated to increase the chance of transformation.
Seeds obtained from these plants were suspended in 0.1% agarose and
placed on % MS medium with 0.8% agarose, 200mg L1 kanamycin and 100mg L1
timentin, kept at 4°C for 48 hr, and then transferred to a lighted culture room at
25°C. Putative transformants (green seedlings) were transferred to the
greenhouse. They were planted in soli (Sunshine, Professional blend) and covered
with plastic wrap for 7 days, and grown to matunty. The light intensity was 800
Lumen m2 (16 hr light /8 hr dark cycle) and temperature setting was 72°C.
Watering was done twice a week with % strength Peters 20-20-20 solution.
T1
seeds were harvested and seeds were germinated on selective medium (with
200mg L1 kanamycin, 100mg L1 timentin) to identify successfully transformed
42
progeny. The numbers of green and white seedlings were determined and green
seedlings were placed in the greenhouse to obtain T2 seed.
PCR and Southern analysis
PCR was used to detect the presence of T-DNA insertion in transgenic
plants. DNA was extracted from tobacco and Arabidopsis leaf pieces (100mg) by
the CTAB (hexadecyltnmethylammonium bromide) method (Doyle and Doyle,
1990). PCR primers specific to the GUS gene were used. A PTC200 thermal
cycler from MJ Research was used for PCR. The PCR conditions were: 1 cycle of
95°C for 3 mm; 32 cycles of 95°C for 30 sec; 68°C for 3 mm and I cycle of 68°C for
5 mm. After the reaction, DNA was kept at 4°C until loaded on a 1.1% agarose gel.
For Southern blot analyses, DNA was extracted from 2g of tobacco leaf
tissue or 0.5g Arabidopsis leaf tissue using he CTAB method (Doyle and Doyle,
1990). The amounts of polysacchandes were reduced by placing the plants in the
dark room at room temperature for 48 hr before DNA extraction. DNA of tobacco
(15p.g) and Arabidopsis (5jig) was digested with Xmnl,
Hincilll,
or BgIll. DNA was
separated on a 1.1 % agarose gel. The gel was soaked in 0.25M HCI for 30 mm, in
denaturation buffer (0.5M NaOH and I M NaCI) for 30 mm, and neutralization
buffer (0.5M Tris buffer (pH 7.4) containing 3M NaCI) for 30 mm. The DNA was
transferred to a nylon Zeta-probe membrane (Bio-Rad) by capillary reaction. The
blotted membrane was washed with 2x SSC and then baked at 80°C for 1 hr to fix
DNA onto the membrane. The 32P-labeled probe, covering the GUS gene, was
43
synthesized using an Oligolabelling kit (Amersham Biosciences) and purified using
a Micro Bio-spin 30 chromatography column (Bio-Rad). Pre-hybridization and
hybndization procedures were performed according to the Zeta-Probe GT
Membrane protocol. A Robbins Scientific Model 1000 hybridization incubator at
60°C was used for both steps. After washing with 2x SSC and 2x SSC containing
0.1% SDS, hybridizations were visualized by autoradiography on Hyperfilm-MP
(Amersham).
GUS assay
Tissues were obtained from transformed tobacco and Arabidopsis plants to
determine GUS expression. The GUS reaction mixture contained 0.1M Na
phosphate buffer (pH 7), 5mM K3Fe(CN)6, 5mM K4Fe(CN)6, 0.O1M EDTA pH 8,
0.01% triton X, and 1mg 5-bromo-4-chloro--indolyl-3-D-glucuronic acid (X-gluc)
in lOpi dimethylformamide (DMF) per imI reaction. Tissues were infiltrated in the
GUS reaction mixture for 1 hr under vacuum (-80 ba), after which they were left
overnight at 37°C. Chlorophyll was eliminated with 95% ethanol.
To better describe the sample age and developmental stage, tobacco
leaves were numbered consecutively with the bottom (first emerging) leaf as the
number 1 (see Fig. 3.1A for illustration). Tobacco flowers were classified into
seven stages according to size and age with number 1 being the immature flower
bud (see Fig. 3.1B).
44
A
Shoot tip
Stem sample
Young seeds
inside
4' .;.
Stagel
2
3
4
5
6
7
/
Fully matured flower
Figure. 3.1. Diagrammatic illustration of tobacco samples used for GUS
assay. (A) Leaf and stem samples. (B) Stages of tobacco flowers from very
young buds (1) to seed pods (7).
45
Whole seedlings of Arabidopsis and vegetative parts of mature plants were
sampled to determine Gus expression.
Induction conditions
As a first step to determine if promoter activity can be induced by plant
hormones, leaf discs of pZOX1::GUS and wild-type tobacco were incubated in
MS medium or H20 with 1O.tM zeatin, 10pM N6-benzyladenine (BAP)'or 10pM
naphthaleneacetic acid
(NAA).
All
growth
regulators
were
dissolved
1/2
a
in
dimethylsulfoxide (DMSO) and 1OpL was added to 20m1 medium. Control tissues
were treated with the same amount of DMSQ added to % MS medium. For longer
experiments, 2-week-old tobacco
seedlings grown on MS medium were
transferred to MS medium with 10pM BAP, 10pM zeatin, 10pM NAA, or no growth
regulators.
46
Results
Genetic characterization of transformants
Transgenic tobacco plants containing each of the three constructs were
obtained. These included eleven families each of pZOXI::GUS and SP::GUS, and
nine families of noP::GUS control plants. For Arabidopsis, ten families of
pZOX1::GUS, eleven of SP::GUS, and five of noP::GUS were generated. T1
progenies were tested for kanamycin resistance. The segregation data revealed
families containing one, two, or more unlinked inserts (Tables 3.1 and 3.2).
PCR was used to detect the presence of the GUS gene in kanamycinresistant T1 plants. All resistant plants yielded a POR product of the expected size
whereas the non-transformed and water control samples were negative (Figure
3.2).
Southern analyses also confirmed the presence of inserts (GUS gene) in
the selected
T1
progenies. As tobacco genomic DNA was digested with enzymes
that do not cut the GUS gene
bands
roughly
correlates
(Hindlll, BgIll,
with the
and Xmnl), the number of hybridizing
number of inserts. All pZOX1::GUS
transformants probed were positive. Multiple inserts of pZOX1::GUS were
detected (shown as multiple bands) in some lines such as #1, 2, 3 and 4 (Figure
3.3), compatible with the segregation data for kanamycin resistance (Table 3.1).
Of the two lines that segregated 3:1, pZOXI::GUS #5 and pZOXI::GUS #6, the
latter had multiple Southern-positive bands, which may represent inserts in
tandem. Similarly, Arabidopsis pZOX1::GUS #1 also showed more than one band
on the Southern blot, even though the segregation ratio indicates a single locus.
Table 3.1. Segregation of T1 transgenic tobacco for kanamycin resistance.
Kan r: Kanamycin resistant seedlings.
Kan s: Kanamycin sensitive seedlings.
Kan r
Kan s
expected segregation
X2
probability
#1
83
2
63:1
0.35
0.5<p<O.75
#2
#3
#4
#5
#6
#7
#8
#9
#10
71
1
63:1
0.01
p>O.9
87
2
63:1
0.27
O.5<p<O.75
42
47
20
57
77
65
32
16
3:1
0.21
O.5<p<O.75
16
3:1
0.01
p>0.9
2
2
15:1
O.5<p<O.7S
O.25<p<O.5
O.Ol<p<O.O25
O.25<p<O.5
pZOX1::GUS
SP::GUS
T1
T1
#1
#2
#3
#4
#5
#6
noP::GUS
#1
#2
#3
#4
#5
T1
12
3:1
6
15:1
0.30
0.82
6.30
0.59
2
15:1
0.01
p>0.9
Kan r
42
45
40
83
Kan S
expected segregation
X2
probability
72
54
17
3:1
Kan r
28
15:1
19
3:1
1.23
O.25<p<O.5
20
3:1
19
3:1
0.25<p<0.5
O.l<p<O.25
14
3:1
1.15
1.63
5.78
0.01<p<O.O25
24
3:1
0.00
0.04
O.75<p<O.9
Kan
expected segregation
X2
0
15:1
3:1
39
27
35
2
0
15:1
58
0
63:1
1.87
0.00
0.13
1.80
0.24
106
S
15:1
p>0.9
probability
O.l<p<O.25
p>0.9
O.5<p<0.75
0.1<p<0.25
O.5<p<0.75
Table 3.2. Segregation of T1 generation of transgenic
For kanamycin resistance.
Kan r: Kanamycin resistant seedlings.
Kan s: Kanamycin sensitive seedlings.
Kari r
pZOXI::GUS Ii
Kan
S
Arabidopsis
expected segregation
X2
probability
O.25<p<O.5
05<p<O.75
#1
25
12
3:1
109
#2
#3
#4
#5
#6
9
1
15:1
0.24
37
12
3:1
0.01
p>0.9
30
6
3:1
O.1<p<O.25
8
1
15:1
10
4
3:1
1.33
0.11
0.10
0
15:1
0.80
O.25<p<0.5
#7
12
SP::GUS T
Kan r
#1
30
24
13
#2
#3
#4
0.5<p<O.75
p0.75
expected segregation
X2
probability
6
3:1
1.33
0.1<p<0.25
8
3:1
6
3:1
0.00
0.44
1.33
0.03
p>0.9
0.5<p<0.75
0.l<p<O.25
O.75<p<09
Kan
S
20
0
15:1
#5
8
3
3:1
noP::GUS Ii
Kan r
expected segregation
X2
probability
#1
2
2
31
O.l<p<025
#2
15
1
15 1
1.33
0.00
Kan
S
p>0,9
5.00()
2,00()
5()
I
2
3
I
5
(,
7
)
10
II
12
Figure 3.2. POR products obtained from transgenic tobacco using primers
based on the GUS gene sequence.
Lane 1: Molecular marker. Lane 2: DDW. Lane 3: Wild type #1. Lane 4: Wild
type #2. Lane 5: 35S::GUS control. Lane 6: pZOX1::GUS #1. Lane 7:
pZOX1::GUS #2. Lane 8: pZOX1::GUS #3. Lane 9: pZOX1::GUS #4.
LanelO: pZOX1::GUS#5. Lane 11,12: pZOXI::GUS#6.
49
lo5Obp
1
2
3
4
5 6
7
8
9
10 1112 13 14 15 16 17 18 19 20 21 2223 24
1650hp
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Figure 3.3. Southern blots of transgenic tobacco and Arabidopsis probed
with GUS. 1: GUS from pBISN1. 3: GUS from pBISN1. 4: Molecular
marker. 6-10: pZOX1::GUS tobacco #145 (cutwith Bglll). 11,12:
pZOX1::GUS tobacco #6 (cut with BgIll). 14: 35S::GUS tobacco positive
control (cut with BgIll). 16-20: pZOX1::GUS tobacco #145 (cut with
Hindlll). 21,22: pZOX1::GUS tobacco #6 (cut with HindUl). 24: 35S::GUS
tobacco (cut with Hindlll). 25: GUS from pBISN1. 27: GUS from pBISN1.
28: Molecular marker. 30-34: pZOX1::GUS tobacco #1-#5 (cut with Xmnl).
35,36: pZOX1:.GUS tobacco #6 (cut with Xmnl). 38: 35S::GUS tobacco
positive control (cut with Xmnl). 40,41: pZOX1::GUS Arabidopsis #1 -#2
(cut with XmnI). 42: noP::GUS Arabidopsis (cut with Xmnl). 43: SP::GUS
Arabidopsis (cut with Xmnl).
GUS expression of transgenic plants
pZOX1-mediated GUS activity was observed in transgenic tobacco.
Immature seeds containing the pZOX1::GUS construct were GUS-positive, with
blue pattern coinciding with the location of endosperm, while seeds from control
50
transformants harbonng noP::GUS and the wild type were negative. This result
confirms that the pZOXI fragment has similar properties as in its native host,
activity in seed and endosperm (Fig. 3.4). Expression of pZOX1::GUS in leaves of
tobacco was confined to trichomes, and was especially evident in trichomes of
young leaves (Fig. 3.5). For example, GUS expression in pZOX1::GUS
T1
#3 was
analyzed at the 24-leaf stage. The tnchomes of leaves 15, 18, 20, and 24
developed blue color, while only few of those on leaf 10 were blue and none on the
lower leaves. Among the 10 pZOXI::GUS transformed tobacco, three lines (#1,
#2, #3) showed the most pronounced and consistent GUS activity and in all cases,
blue stain is observed only in trichomes. Other pZOXI::GUS lines (#6, #8, #9 and
#10) were positive in some tests but not others. As expected, the SP::GUS plants
developed GUS stain in leaves (Fig. 3.5) and other tissues. Neither the noP::GUS
nor wild-type controls showed GUS expression in trichomes or other leaf cells.
Stem tissues of tobacco showed GUS expression in hairs (Fig. 3.6). Also in
this case the location of the hairs seemed to have an effect. For instance, hairs of
the upper stem of pZOX1::GUS T #3 between leaves 18 and 19 stained uniformly
blue, while only some of those between leaves 3 and 4 and right under the stem
tip were positive. No GUS activity was observed on stems of noP::GUS and wildtype tobacco.
Among reproductive tissues of transgenic tobacco, the pollen, stigma, and
anthers showed some GUS expression; however, the controls (noP::GUS and wild
type) were also positive, indicating the presence of endogenous J3-glucuronidase
activity. These tissues are not appropriate samples to assess promoter activity
using GUS as the reporter gene.
5'
Figure 3.4. Young seeds of pZOX1::GUS tobacco expressing GUS.
(A)Young seeds of pZOX1::GUS T1#1 tobacco (B) Strong GUS
expression in endosperm in young seeds of pZOX1::GUS T#3 tobacco.
Figure 3.5. Transgenic tobacco leaf trichomes expressing GUS.
(A)(C) 35S::GUS tobacco trichomes on the leaf.
(B)(D) pZOX1::GUS Ti #3 transgenic tobacco leaf trichomes.
52
Figure 3.6. Hairs on stems of pZOX1::GUS tobacco showing GUS activity.
Interestingly, and in contrast to tobacco,
Arabidopsis
pZOX1::GUS
transformants did not show any GUS activity in leaves, trichomes, or stem hairs. In
limited tests, the seeds were also negative; however, additional sampling is
needed to be certain of the inactivity of the ZOX1 promoter in this model plant.
Induction of pZOX1 promoter by external factors
To determine if pZOX1::GUS can be induced by hormones, leaf discs were
incubated in % MS medium or distilled water containing cytokinins and auxins. For
these experiments, old leaves of pZOX1::GUS tobacco, not normally showing
53
GUS expression, were used. Cytokinins seemed to induce GUS expression in
pZOX1::GUS. For instance, trichomes of leaf 4 of pZOX1::GUS #3, not
expressing GUS before the experiment, showed GUS stain after incubation with
1 Op.M BAP or 1 Op.M zeatin for 5 days. The color was more intense on BAP than
zeatin.
In order to expose tissues for a longer time to growth regulators, seeds of
pZOXI::GUS #1, #2 and #3 were germinated on MS medium and 2-week-old
seedling were transferred to fresh medium with 1OiM BAP, lOj.tM zeatin, lOp.M
NAA, or no growth regulators. GUS activity was induced by growth regulators in all
three lines but at different times. Leaves of line #1 showed clear GUS stain in
veins and some of the tnchomes after only 3 days on BAP, zeatin or NAA. Line #2
showed GUS expression after 3 weeks on all growth regulators. Line #3 showed
GUS-positive trichomes after 3 days on zeatin or I week on BAP or NAA. The
seedlings grown on medium without growth regulators never showed any GUS
stain in trichomes and only occasionally in veins. The SP::GUS control had strong
GUS expression throughout the culture period while the wild type did not.
Discussion
0-Glycosylation of cytokinins occurs in many plant tissues and the process
is likely related to transient storage and transport. 0-Glucosides are resistant to
cytokinin oxidase attack and cytokinin activity can be restored through reversion to
cytokinin aglycones by 3-glucosidases. The level of 0-glycosylation, therefore, will
54
affect the active cytokinin pooi in cells and tissues as well as the distribution of
cytokinins between organs of the whole plant. The cloning of zeatin 0glycosylation genes such as ZOG1 and ZOX1 provided the tool to examine the
precise regulation of 0-glycosylation. ZOG1 and ZOX1 have similar expression
patterns based on Northern analyses and thus the promoters of the two genes are
likely to be under similar regulatory controls. As transformation of
Phaseolus is
extremely difficult, the transgenic approach to study promoter activity began with
model plants, tobacco and Arabidopsis.
The choice of a 1.2-kb upstream fragment of ZOX1 fused to the GUS
reporter gene as described in this chapter appears to be suitable in obtaining
preliminary data on the property of the promoters. The 1.2-kb fragment should
contain the promoter of ZOXI since the pattern of pZOXI::GUS expression in
transgenic tobacco seed, with high expression in endosperm, is the same as
observed in the native host, P. vulgaris Further paring of this fragment should
allow the identification of essential and necsssary sequences of the promoter.
In vegetative tissues, pZOX1 mediates reporter expression only in
tnchomes and stem hairs, both originated from epidermal cells. The differentiation
of epidermal cells into these specialized structures usually involves a change of
the direction of cell division and endomitosis (of the nucleus) resulting in cells with
polyploid DNA contents. The differentiation of trichomes in Arabidopsis is better
understood and more than 20 genes regulate the formation and maturation of
trichomes (Marks, 1997). Although no comparable information on tobacco
trichome formation is available, it is reasonable to assume that key components
are similar. The fact that transcription is driven by pZOX1 in tobacco trichomes and
55
stem hairs would suggest that the promoter activity is tied to either endomitosis or
a switch in the polarity of cell division. The former is compatible with activity of
pZOX1 in bean and transgenic tobacco endosperm, tissue with endo-duplicated
nuclei through multiple rounds of DNA replication. Trichomes and hairs can be
unicellular (as in Arabidopsis) or multi-cellular (as in tobacco) and are
metabolically active. It is possible that expression of cytokinin 0-glycosylation
genes in such tissues can transiently reduce active cytokinins to accommodate
alternating cycles of rapid duplication and rest. However, these hypotheses remain
to be confirmed by more detailed experiments.
It is not known why pZOX1::GUS activity was observed only in tobacco but
not in Arabidopsis. A possible explanation is that key transcription factor(s)
required to activate the pZOX1 promoter are absent in Arabidopsis. This
suggestion is supported indirectly by the observation that, based on sequence
homology searches involving the complete Arabidopsis genome, no genes with
high homology to ZOG1 or ZOXI are presett in Arabidopsis. Thus, cytokinin 0glycosylation, if it occurs in Arabidopsis, must be mediated by somewhat different
enzymes. The genes encoding such enzymes are likely to have promoters
responding to different signals. The differential expression of pZOXI::GUS in
these two model plants provides an opportunity to isolate and clone speciesspecific transcription factors.
Induction of promoter activity by internal and external signals was explored
to a limited extent. It appears that pZOX1::GUS expression can be activated by
cytokinins and auxins, especially in trichomes of leaves usually no longer
expressing GUS. The experiment involving incubation of leaf discs for a short
56
period of time (up to 5 days) was easier to interpret since trichome ceUs exposed
to zeatin and BAP were clearly GUS active. However, it is not yet clear if the action
is related to induction of promoter activity in existing cells or cytokinins inducing a
new round of cell division. Further experiments involving mapping of trichome
location prior to incubation will allow distinction between these two possibilities.
Experiments with cultured seedlings gave ambiguous results since the timing of
GUS expression was not uniform. Better protocols may need to be devised to
allow the study of gene induction over longer periods of growth.
Certain transgenic tobacco families (pZOX1::GUS #1, #2, and #3) had
more consistent and stronger pZOX1-mediated GUS expression. These families
contained multiple inserts based on segregation for kanamycin resistance as well
as Southern analyses. Therefore, it is possible that the ZOXI promoter is not
strongly transcribed in tobacco and multiple copies are requiredfor activation of
the reporter gene.
The results described in this chapterprovide key insights into the
transcriptional regulation of zeatin 0-glycosylation. Although preliminary in nature,
the findings suggest possibilities for new approaches, and the transgenic materials
generated are essential for further studies of regulatory pathways controlling levels
of active cytokinins.
References
Bassil NV, Mok DWS, and Mok MC (1993) Partial punfication of a cis-trans
isomerase of zeatin from immature seed of Phaselous vulgaris L., Plant Physiol
102: 867-872.
Bechtold N, Ellis J, and Pelletier G (1993) In Planfa Agmbacterium-mediated gene
transfer by infiltration of adult Arabidopsis thaliana plants, Life Sciences 316: 11941199.
Clough Si and Bent AF (1998) Floral dip: a simplified method for Agrobactenmmediated transformation of Arabidopsis f/ia/lana, Plant J 16: 735-743.
Dixon SC, Martin RC, Mok MC, Shaw G, and Mok DWS (1989) Zeatin
glycosylation enzymes in Phaselous: isolation of 0-glucosyltransferase from P.
lunatus and companson to O-xylosyltransferase from P. vulgaris, Plant Physiol 90:
1316-1321.
Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue, Focus 12:
13-15.
Hofgen R and Wiltmitzer L (1988) Storage of competent cells for Agrobacterium
transformation, Nucleic Acids Res 6: 9877.
Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, and Fraley RI (1988)
Leaf disc transformation, In: Gelvin SB and Schilperoort (eds.) Plant molecular
biology manual A5, pp. 1-9, Kluwer Academic Publishers, Dordrecht.
Marks DM (1997) Molecular genetic anaIysi of tnthome development in
Arabidopsis, Annu. Rev. Plant Physiol. Plant Mol 6101 48: 137-163.
Martin RC, Mok MC, and Mok DWS (1993) Cytolocalization of zeatin 0xylosyltransf erase in Phaselous, Proc NatI Acad Sci 90: 953-957.
Martin RC, Mok MC, and Mok DWS (1999a) Isolation of a cytokinin gene, ZOG1,
encoding zeatin 0-glucosyltransferase of Phaseo/us /unatus, Proc NatI Aced Sd
USA 96: 284-289.
Martin RC, Mok MC, and Mok DWS (1999b) A gene encoding the cytokinin
enzyme zeatin 0-xylosyltransferase of Phaseo/us vulgaris, Plant Physiol 120:
553-557.
Turner JE, Mok DWS, Mok MC, and Shaw G (1987) Isolation and partial
purification of an enzyme catalyzing the formation of 0-xylosylzeatin in Phaselous
vulgans embryos, Proc NatI Aced Sci USA 84: 3714-3717.
58
CHAPTER 4
GENERAL CONCLUSION
Zeatin and its derivatives are major components of naturally occurring
cytokinins. t,ns-Zeatin has high biological activity and the free base and its
denvatives have been the focus of cytokinin biology. Finding substantial levels of
cis-zeatin in specialized organs and the isolation of genes such as cisZOGs in
maize redirect attention to this group of cytokinins. The properties of the cisZOG
enzyme described in the present study indicate clearly a preference for cis- over
trans-zeatin, suggesting the existence of cis-specific metabolic pathways. Maize
and other monocots seem to have relatively high levels of cis-zeatin and
derivatives, and 0-glucosylation may be important in regulating appropriate levels
of the active forms. Up or down regulating cisZOG genes may provide further
clues to the function of cis-compounds in deielopment.
The regulation of cytokinin 0-glycosylation genes is being examined. The
results described in this study using the pZOX1 promoter suggest that gene
expression is likely to be associated with rapid cell division and changes in cell
polarity. As endosperm and tnchomes development involves endoduplication, an
inference is that 0-glycosylation may be related to transient and reversible
reduction of active cytokinins during rapid nuclear division to accommodate
alternating cycles of duplication and rest. The preliminary findings and transgenic
plants are important guides and tools for further testing of this hypothesis.
59
Armstrong DJ (1994) Cytokinin oxidase and the regulation of cytokinin
degradation. In: Mok DWS and Mok MC (eds.) Cytokinins- chemistry, activity, and
function, pp. 139-154, CRC Press, Boca Raton.
Astot C, Dolezal K, NordstrOm, Wang Q, Kunkel T, Moritz T, Chua NH, and
Sandberg G (2000) An alternative cytokinin biosynthesis pathway, Proc Nati Acad
Sd USA 97: 14778-14783.
Barry GF, Rogers SG, Fraley RT, and Brand L (1984) Identification of a cloned
cytokinin biosynthetic gene, Proc NatI Acad Sci USA 81: 4776-4780.
Bassil NV, Mok DWS, and Mok MC (1993) Partial purification of a cis-trans
isomerase of zeatin from immature seed of Phaselous Vulgaris L., Plant Physiol
102: 867-872.
Bechtold N, Ellis J, and Pelletier G (1993) In Planta Agmbactei'ium-mediated gene
transfer by infiltration of adult Arabidopsis thaliana plants, Life Sciences 316:11941199.
Bilyeu KD, Cole JL, Laskey JG, RiekhofWR, Esparza TJ et al. (2001) Molecular
and biochemical characterizaion of Maize, Plant Physiol, 125: 378-386.
Brzobohat B, Moore I, Knstofferson P, Bakó L, Campos N et al. (1993) Release
of active cytokinin by a fl-glucosidase localized to the maize root meristem,
Science 262: 1051-1054.
Buchmann I, Mamer FJ, SthrOder G, Waffeischmidt S, and SchrOder J (1985)
Tumor genes in plants: T-DNA encoded cytokinin biosynthesis, EMBO J 4: 853859.
Campos N, Bakô L, Feldwisth J, Schell J, and Palme K (1992) A protein from
maize labelled with azido-IAA has novel /3-glucosidase activity, Plant J 2: 675-684.
Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterimmediated transformation of Arabidopsis thaliana, Plant J 16: 735-743.
Davies PJ (eds.)(1995) Plant hormones; physiology, biochemistry, and molecular
biology, 2nd edition, Kluwer Academic Publishers, Dordrecht.
Dixon SC, Martin RC, Mok MC, Shaw G, and Mok DWS (1989) Zeatin
glycosylation enzymes in Phaselous: isolation of 0-glucosyltransferase from P.
Iunatus and comparison to O-xylosyltransferase from P. vulgans, Plant Physiol 90:
1316-1321.
60
Dobrev P, Motyka V, Gaudinová A, Malbeck J, Trávniková A, Kaminek M, and
Vanková R (2002) Transient accumulation of cis- and trns-zeatin type cytokinins
and its relations to cytokinin oxidase activity during cell cycle of synchronized
tobacco cells, Plant Physiol Biochem 40: 333-337.
Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue, Focus 12:
13-15.
Durand R and Durand B (1994) Cytokinins and reproductive organogenesis in
Mercurialis, In: Mok DWS and Mok MC (eds.) Cytokinins-chemistry, activity, and
function, pp. 295-304, CRC Press, Boca Raton.
Einset JW (1984) Conversion of N6-isopentenyladenine to zeatin by Actinidia
tissues, Biochem Biophys Res Commun 124: 470-474.
Emery RJN, Leport L, Barton JE, Turner NC, and Atkins CA (1998) cis-Isomers of
cytokinins predominate in chickpea seeds throughout their development, Plant
Physiol 117: 1515-1523.
Emery RJN, Ma Q, and Atkins CA (2000) The forms and sources of cytokinins in
developing white lupine seeds and fruits, Plant Physiol 123: 1593-1604.
Entsch B, Parker CW, Letham OS, and Summons RE (1979) Preparation and
characterization using high-performance liquid chromatography of an enzyme
forming glucosides of cytokinins, Biochem Biophys Acta 570: 124-139.
Entsch B and Letham DS (1979) Enzymic glycosylation of the cytokinin, 6-benzylaminopurine, Plant Sd Left 14: 205-2 12.
Entsch B, Letham OS, Parker CW, and Sunmons RE (1979) preparation and
characterization, using high performance liquid chromatography, of an enzyme
forming glucosides of cytokinins, Biochem Biophys Acta 570: 124-139.
Entsch B, Letham DS, Parker CW, Summons RE, and Gollnow BI (1980)
Metabolites of cytokinins, In: Skoog F (eds.)(1979) Plant growth substances,
Biochem Biophys Acta 570: 124-139.
Gan S and Amasino RM (1996) Inhibition of leaf senescence by autoregulated
Production of Cytokinin, Science 270(22): 1986-1988.
Gersani M and Kende H (1982) Studies on cytokinin-stimulated translocation in
isolated bean leaves, J Plant Growth Regul 1: 161-171.
Gray J, Wang J, and Gelvin SB (1992) Mutation of the miaA gene of
Agrobactenum tumefaciens results in reduced vir gene expression, J Bacteriol
174: 1086-1098.
61
Guivarc'h A, Rembur J, Goetz M, Roitsch T, Noin M, Schmufling T, and Chriqui D
(2002) Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum
L. cv. SRI) axillary buds establishes a role for cytokinins in tuberization and sink
formation, J Experimental Botany 53 (369): 621-629.
Hofgen R and Willmitzer L (1988) Storage of competent cells for Agrobacterium
transformation, Nucleic Acids Resl6: 9877.
Horsch RB, Fry J, Hoffmann N, NeidermeyerJ, Rogers SG, and Fraley RT (1988)
Leaf disc transformation, In: Gelvin SB and Schilperoort (eds.) Plant molecular
biology manual A5, pp. 1-9, Kluwer Academic Publishers, Dordrecht.
Houba-Henn n, Pethe C, d'AlayerJ, Laloue M (1999) Cytokinin oxidase from Zea
mays: puriifcation, cDNA cloning and expression in moss protoplasts, Plant J
17:615-626.
Jefferson RA, Kavanagh TA, and Bevan MW (1987) GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants,
EMBO J 6: 3901-3907.
Kaiss-Chapman RW and Moms RO (1977) T,ns-zeatin in culture filtrates of
Agrobacterium tumefacien, Biochem Biophys Res Comm 76: 453-459.
Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as
Dimethylallyl Diphosphate: ATP/ADP isopentenyltransferases, Plant Cell Physiol
42(7): 1-111.
Letham DS, Wilson MM, Parker CW, Jenkins ID, MacLeod JK, and Summons RE
(1975) Regulators of cell division in plant tissues. XXII. The identity of an unusual
metabolite of 6-benzylaminopunne, Biochem Biophys Acta 399: 61-70.
Letham DS, Palni LMS, Tao GQ, Gollnow BI, and Bates CM (1983) Regulators of
cell division in plant tissues. XXIX. The activities of cytokinin glucosides and
alanine conjugates in cytokinin bioassay, Plant Growth Regul 2:103-115.
Letham DS and Zhang R (1989) cytokinin translocation and metabolism in lupin
species. II. New nucleotide metabolites of cytokinins, Plant Sci 64: 161-165.
Letham DS (1994) cytokinins as phytohormones-sites of biosynthesis,
translocation, and function of translocated cytokinin, In: Mok DWS and Mok MC
(eds.) Cytokinins- chemistry, activity, and Function, pp. 57-80, CRC Press, Boca
Raton.
62
Letham YY (1988) Plant senescence processes and free radicals, Free Radical
Biology and Medicine 5: 39-49.
Li Y, Baldauf S, Lim E-K, and Bowles DJ (2001) Phylogenetic analysis of the UDPglycosyltransferase multigene family of Arabidopsis thallana, J Biol Chem
276: 4338-4343.
Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of
isoprenoid biosynthesis in plants, Annu Rev Plant Physiol Plant Mol Biol 50: 4765.
Louis J-P, Augur C, and Teller G (1990) Cytokinins and differentiation processes in
Mercurialis annua, Plant Physiol 94: 1535-1541.
Marks DM (1997) Molecular genetic analysis of trichome development in
Arabidopsis, Annu. Rev. Plant Physiol. Plant Mol Biol 48: 137-163.
Martin RC, Mok MC, Shaw G, and Mok DWS (1989) An enzyme mediating the
conversion of zeatin to dihydrozeatin in Phaselous embryos, Plant Physiol 90:
1630-1635.
Martin RC, Mok MC, and Mok DWS (1993) Cytolocalization of zeatin 0xylosyltransferase in Phaseious, Proc NatI Aced Sci 90: 953-957.
Martin RC, Mok MC, and Mok DWS (1999a) Isolation of cytokinin gene, ZOGI,
encoding zeatin 0-glucosyltransferase of Phaselous lunatus, Proc NatI Aced Sci
USA 96: 284-289.
Martin RC, Mok MC, and Mok DWS (1999b)A gene encoding the cytokinin
Enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris, Plant Physiol 120: 553558.
Martin RC, Mok MC, Habben JE, and Mok DWS (2001) A maize cytokinin gene
encoding an 0-glucosyltransferase specific to cis-zeatin, Proc NatI Aced Sd USA
98(10): 5922-5926.
Mauk CS and Langille AR (1978) Physiology of tuberization in Solanum tuberosum
L. Cis-zeatin riboside in the potato plant: its identification and changes in
endogenous levels as influenced by temperature and photoperiod, Plant Physiol
62: 438-442.
Miller CO, Skoog F, von Saltza MH, and strong M (1955) Kinetin, a cell division
factor from deoxynbonucleic acid, JAm Chem Soc 77: 1329-1334.
Mok MC and Mok DWS (1987) metabolism of 14C-zeatin in Phaselous embryos.
Occurrence of O-xylosyldihydrozeatin and its ribonucleoside, Plant Physiol 84:
596-599.
63
Mok MC (1994) Cytokinins and plant development - an overview, In: Mok DWS
and Mok MC (eds.) Cytokinins- chemistry, activity, and function, pp. 155-166, CRC
Press, Boca Raton.
Mok DWS and Mok MC (eds.)(1994) Cytokinins- chemistry, activity, and function,
CRC press, Boca Raton.
Mok MC and Mok DWS (2001) Cytokinin metabolism and action, Annu Rev Plant
Physiol Plant Mo! Biol 52: 89-118.
Moms RO, Bilyeu KD, Laskey JG, and Cheikh NN (1999) Isolation of a gene
encodong a glycosylated cytokinin oxidase from maize. Biochem Biophys Res
Commun 255: 328-333.
Nielsen TH and Ulvskov P (1992) Cytokinins and leaf development in sweet
pepper (Capsicum annuum L.) II. Sink metabolism in relation to cytokininpromoted leaf expansion, Planta 188: 78-84.
Paces V, Werstiuk F, and Hall RH (1971) conversion of N6-(A2isopentenyl)adenosine to adenosine by enzyme activity in tobacco tissue, Plant
Physiol 48: 775-778.
Parker CW, Badenoch-Jones J, and Letham DS (1989) Radioimmunoassay for
quantifying the cytokinins cis-zeatin and cis-zeatin riboside and its application to
xylem sap samples, Plant Growth Regul 8: 93-105.
Richmond A and Lang A (1957) Effect of kinetin on protein content and survival of
detached Xanthium leaves, Science 125: 6EJ-651.
Schmitz RY, Skoog F, Playtis AJ, and Leonard NJ (1972) Cytokinins: synthesis
and biological activity of geometric and position isomers of zeatin, Plant Physiol
50: 702-705.
SchmUlling T, Beinsberger S, De Greef J, Schell J, Van Onckelen H, and Spena A
(1989) Construction of a heat inducible chimaeric gene to increase the cytokinin
content in transgenic plant tissue, FEBS Lett 249: 401-406.
Skoog F and Miller CO (1957) Chemical regulation of growth and organ formation
in plant tissues cultured in vifm, Symp Soc ExpI Biol 11: 118-131.
Skoog F and Armstrong DJ (1970) Cytokinins, Annu Rev Plant Physiol 21: 359384.
Smigocki AC and Owens LD (1988) Cytokinin gene fused with a strong promoter
enhances shoot organogenesis and zeatin levels in transformed plant cell, Proc
Nat! Acad Sci USA 85: 51 31-51 35.
64
Stmad M (1997) The aromatic cytokinins, Plant Physiol 101: 674-688.
Suttle JC and Banowetz GM (2000) Changes in cis-zeatin and cis-zeatin riboside
levels and biological activity during tuber dormancy, Physiol Plant 109: 68-74.
Takagi M, Yokota T, Murofushi N, Ota Y, and Takahashi N (1985) Fluctuation of
endogenous cytokinin contents in rice during its life cycle - quantification of
cytokinins by selected ion monitoring using deuterium-labeled internal standards,
Agnc Biol Chem 49: 3271-3277.
Takagi M, Yokota T, Murofushi N, Saka H, and Takahashi N (1989) Quantitative
changes of free-base, riboside, ribotide and glucoside cytokinins in developing rice
grains, Plant Growth Regul 8: 349-364.
Takei K, Sakakibara H, and Sugiyama T (2001) Identification of genes encoding
adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis
thaliana, J Biol Chem 276: 26405-26410.
Takei K, Takahashi T, Sugiyama T, Yamaya T, and Sakakibara H (2002) Multiple
routes communicating nitrogen availability from roots to shoots: a signal
transduction pathway mediated by cytokinin, J. Experimental Botany 53 (370):
971-977.
Tantikanjana T, Yong JWH, Letham DS, Griffith M, Hussain M, Ljubg K, Sandberg
G, and Sundaresan V (2001) Control of axillary bud initiation and shoot
architecture in Arabidopsis through the SUPERSHOOT gene, Genes and
Development 15(12): 1577-1588.
is a direct precursor of cytokinin
Taya Y, Tanak Y, and Nishimura S (1978)
in Dictyostelium discoidum, Nature 271(541): 545-547.
Thimann Ky (1987) Plant senescence: A proposed integration of the constituent
processes, In: Thomson WW, Nothnagel EA, and Huffaker (eds.) Plant
senescence: Its biochemistry and Physiology, pp. 1-19, American society of plant
physiologists, Rockville, Md.
Thomson JE, Legge RL, and Barber TF (1987) The role of free radicals in
senescence and wounding, New Physiologist 105: 317-344.
Turner JE, Mok DWS, Mok MC, and Shaw G (1987) Isolation and partial
purification of an enzyme catalyzing the formation of O-xylosylzeatin in Phaselous
vulgaris embryos, Proc NatI Aced Sci USA 84: 3714-3717.
Van Staden J, Cook E, and Noodén LD (1988) Cytokinins and senescence, In:
Noodén LD and Leopold AC (eds.) Senescence and aging in plants, pp. 28 1-328,
Academic Press, San Diego.
65
Vañková R (1999) Cytokinin glycoconjugates-distnbution, metabolism and
function, In: Strand M, Pea, Beck E (eds.) Advances in Regulation of Plant Growth
and Development, pp. 67-78, Prague, Peres.
Watanabe N, Yokota T, and Takahashi N (1981) Variations in the levels of cis- and
tns-ribosylzeatins and other minor cytokinins during development and growth of
cones of the hop plant, Plant Cell Physiol 22: 489-500.
Werner T, Motyka V, Strnad M, and Schmulling T (2001) Regulation of plant
growth by cytokinin, Proc Nat Acad Sd USA 98(18): 10487-10492.
Zubko E, Adams C, Macháèková I, Malbeck J, Scollan C, and Meyer P (2002)
Activation tagging identifies a gene from Petunia hybrida responsible for the
production of active cytokinins in plants, Plant J 29(6): 797-808.