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Chapter9
Gene Expression Studies1
Lise Jouanin and Gilles Pilate
Introduction
In this chapter, we discuss published research on the
isolation and expression of poplar genes, including
complementary eDNA (eDNA) and genomic DNA of
several characterized genes. The over and under expression of introduced genes in homologous or heterologous host plants is discussed and published results are
presented. Gene expression studies in poplar are now
possible because procedures to transform and regenerate different genotypes are commonly available (reviewed in Jouanin et al. 1993 and this volume). Genes
of plant origin are the focus of this chapter. Sequences of nonplant origin are discussed elsewhere
in this book.
Poplar Gene Isolation
The list of genes from woody plants that have been
isolated, sequenced, and published is relatively small.
Continued effort is directed toward understanding the
metabolic aspects of trees. Poplar is particular! y amenable to gene isolation because of its small genome size
(1.12 picograms DNA/diploid genome), which promotes the production of genomic libraries representative of the complete genome.
1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, LG., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
Among trees, most of the available gene sequences
are from poplar (mainly eDNA sequences). This information is summarized in table 1. Research efforts have
focused on genes: 1) involved in the wounding response
(Bradshaw et al. 1989; Davis et al. 1991; Parsons et al.
1989); 2) involved in lignification (Bugos et al. 1991;
Dumas et al. 1992; Kawai et al. 1993; Osakabe et al.1994,
1995a, b, c; Subramanian et al. 1993; Tsai et al. 1995;
van Doorsselaere et al. 1995a); and 3) encoding storage
proteins (Clausen and Apel1991; Coleman et al. 1992).
In most studies, gene sequences from annual dicotyledonous plants were the basis of probes or polymerase
chain reaction primers used to isolate pop!ar cDNAs
(potato PAL eDNA: Subramanian et al. 1993; tobacco
CAD eDNA: van Doorsselaere et al. 1995a; aspen OMT
primers: Tsai et al. 1995).
Where possible, the poplar eDNA sequences that were
obtained were compared with those of the corresponding genes in annual dicot plants (WIN: Davis et al. 1991;
OMT: Dumas et al. 1992; PAL: Osakabe et al. 1995a). A
high degree of homology was observed between poplar eDNA and annual dicot plant eDNA; frequently
more than 80 percent at the amino acid level. However,
the highest homology was found with sequences originating from other deciduous trees; for example, comparison of CAD and OMT sequences between poplar and
eucalyptus (CAD 80.9 percent, van Doorsselaere et al.
1995a; OMT 84 and 80 percent, Hayakawa et al. 1996).
When present, intron sequences were found in the expected locations, as demonstrated by genes encoding
chitinases (Davis et al. 1991) and anionic peroxidases
(Osakabe et al. 1995b). Overall, poplar genes showed a
closer relationship to dicotyledonous angiosperms than
to gymnosperms or monocotyledons as shown in table 2
(PAL: Osakabe et al. 1995a; CAD: van Doorsselaere et al.
1995a; OMT: Hayakawa et al. 1996).
Most of the studied genes are members of small
multigene families (WIN: Davis et al. 1991; OMT:
Hayakawa et al. 1996; PRX: Osakabe et al. 1995b; PAL:
Osakabe et al. 1995c, CHS: Lurin and Jouanin 1995). In
some cases, they are clustered in the genome (WIN: Davis
et al. 1991; PRX: Osakabe et al. 1995b; PAL: Osakabe et al.
1995c).
65
Section II Transformation and Foreign Gene Expression
Table 1. eDNA and genomic sequences cloned in poplar.
Name
Enzyme
Origin
Poplar type
References
Ptom1
PTOMT
pPCL4
HOMT1 & HOMT2
PAL
cDNA1
PALG1 & G2A
PALG28 & PALG4
POPCAD
(CAD)
WIN3
gWIN3
WIN6& 8
gWIN6,8&X
pPOP1
0-methyl transferase (OMT)
0-methyl transferase (OMT)
0-methyl transferase (OMT)
0-methyl transferase (OMT)
Phenylalanine ammonia-lyase (PAL)
Phenylalanine ammonia-lyase (PAL)
Phenylalanine ammonia-lyase (PAL)
Phenylalanine ammonia-lyase (PAL)
Cinnamyl alcohol dehydrogenase
eDNA
genomic
eDNA
genomic
eDNA
eDNA
genomic
genomic
eDNA
P. tremuloides
P. tremuloides
P. deltoides x P. trichocarpa
P. sieboldii x P. grandidentata
P. deltoides x P. trichocarpa
P. sieboldii x P. grandidentata
P. sieboldii x P. grandidentata
P. sieboldii x P. grandidentata
P. deltoides x P. trichocarpa
Wound-inducible (similar to sporamin
and Kunitz proteinase inhibitor)
Chitinase
Chitinases
Malic enzyme
eDNA
genomic
eDNA
genomic
eDNA
P. trichocarpa x P. deltoides
P. trichocarpa x P. deltoides
P. trichocarpa x P. deltoides
P. trichocarpa x P. deltoides
P. deltoides x P. trichocarpa
samPdxPt
S-adenosyi-L-Methionine synthetase eDNA
DHDPS
BSP
bspA
WS1
pA2, pA3
PRXA1
PRXA3A
PRXA4A
Dihydrodipicolinate synthase
Bark storage protein
Bark storage protein
Xylem ray cell storage protein
Anionic peroxidases
Anionic peroxidase
eDNA
eDNA
genomic
eDNA
eDNA
genomic
P. deltoides x P. trichocarpa
P. deltoides
P. deltoides
P. nigra x P. deltoides
P. sieboldii x P. grandidentata
P. sieboldii x P. grandidentata
Bugos et al. 1991
Tsai et al. 1995
Dumas et al. 1992
Hayakawa et al. 1996
Subramaniam et al. 1993
Osakabe et al. 1995a
Osakabe et al. 1995a
Osakabe et al. 1995c
van Doorsselaere et al.
1995a
Hollick and Gordon 1993
Bradshaw et al. 1989
Parsons et al. 1989
Davis et al. 1991
van Doorsselaere et al.
1991
van Doorsselaere et al.
1993
Vauterin and Jacobs 1994
Coleman et al. 1992
Coleman and Chen 1993
Clausen and Apel 1991
Osakabe et al. 1994
Kawai et al. 1993
Anionic peroxidases
genomic
P. sieboldii x P. grandidentata
Osakabe et al. 1995b
P. deltoides x P. trichocarpa
Table 2. Identity comparison at the amino acid level (%) among the PAL, OMT, and CAD genes of poplar and other plants.
Dicot
Gene
Plant
(%)
PALG2
bean
parsley
bean
parsley
tobacco OMT1
OMT2
eucalyptus
tobacco OMT1
OMT2
eucalyptus
tobacco
alfalfa
(85.9)
(84.7)
(81.5)
(82.8)
(77)
(56)
(84)
(76)
(54)
(80)
(79.8)
(84)
PAL G2
HOMT1
HOMT2
CAD
Monocot
Plant
(%)
References
pine
(66.1)
Osakabe et al. 1995a
pine
(64.2)
Osakabe et al. 1995a
maize
(28)
Hayakawa et al. 1996
maize
(26)
Hayakawa et al. 1996
Gene Expression at the RNA Level
Isolated cDNAs have been used as probes to monitor
gene expression in different tissues and under various
environmental conditions. The most detailed studies have
focused on the systemic accumulation of specific messen-
66
Conifer
Plant
(%)
spruce
(69.2)
van Doorsselaere et al.
1995a
ger RNA (mRNAs) in response to wounding (Parsons et
al. 1989; Bradshaw et al. 1989), seasonal changes and tissue-specific expression of mRNAs encoding bark and xylem ray cell storage proteins (Clausen and Apel 1991;
Coleman .et al. 1992), and genes involved in the phenylpropanmd pathway (PAL: Subramanian et al. 1993;
Osakabeet al. 1995a, c; OMT: Bugos et al. 1991, Hayakawa
et al. 1996; anionic peroxidases: Osaka beet al. 199Sb; CHS:
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
~.
Gene Expression Studies
Lurin and Jouanin 1995). In these studies, mRNA abundance was measured either by northern hybridization or
by reverse transcriptase-polymerase chain reaction. From
these studies, we can infer that gene regulation often occurs at the transcriptional level.
dicot and a woody angiosperm. It may be feasible to study
woody plant gene promoters in a herbaceous plant model
such as tobacco. However, reintroduction of sequences into
the same species is required to study promoter function in
relation to unique structural, metabolic, and developmental features. Currently, there are no reports of poplar promoters reintroduced into poplar. This research field is
expected to develop rapidly.
Poplar Heterologous Promoters
When establishing a genetic transformation procedure
for a specific poplar hybrid,· vectors frequently contain a
neomycin phosphotransferase gene (NPTII), which is a
selectable marker that confers resistance to kanamycin, and
a ~-glucuronidase gene (GUS), which is used as a reporter
of gene expression Qouanin et al. 1993). Binary vectors such
as pBI121 Oefferson et al. 1987) or its derivatives, in which
the NPTII and the GUS genes are placed under control of
the Agrobacterium tumefaciens nopaline synthase (NOS)
promoter and either the 35S or 705 (double enhancer sequen~es) CaMV promoters, function efficiently in poplar
(Leple et al. 1992). Thus, vectors used for dicot transformation are frequently suitable for poplar use.
Reports that illustrate the function of heterologous plant
promoters in poplar include promoters from the potato
proteina.se inhibitor II (PIN2) gene (Klopfenstein et al.
1991 ), the Arabidopsis thaliana acetolacta te synthase (AtALS)
gene (Brasileiro et al. 1992), and the Eucalyptus cinnamyl
alcohol dehydrogenase (EuCAD) gene (Feuillet et al. 1995).
Expression of an associated reporter gene (CAT or GUS)
indicates that regulation is conserved. This is represented
by the wound induction of the PIN2-CAT construct
(Klopfenstein et al. 1991), and the tissue-specificity of the
EuCAD-GU5 construct in relation to lignification {Feuillet
et al. 1995). These studies illustrate the basis of considerable interest in poplar, an easily transformable woody
plant, in which sequences originating from other plant
species have conserved patterns of expression.
Poplar Promoters in
Heterologous Plants
In some cases, the characterization of poplar genomic
sequences has involved promoter isolation. These promoters have been linked to a reporter gene such as GUS to
study their expression pattern in transgenic plants. Two
wound-inducible poplar promoters, WIN3 (Hollick and
Gordon 1993) and WIN6 (Clarke et al. 1994), are also induced upon wounding in tobacco. This demonstrates conservation of expression mechanisms between an annual
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Over and Under Expression of
Poplar Genes in Plants
Of major interest, at the fundamental and applied levels, is the manipulation of gene expression to alter levels
of specific proteins and shift metabolic processes. Such
work in poplar is just emerging. As of this writing, the
only report of over expression of a gene concerns a peroxidase gene in poplar callus (Kajita et al. 1994). The only
report of under expression of a gene is that of a caffeic
acid/5-hydroxyferulic acid 0-methyltransferase (OMT)
gene in poplar stems (van Doorseelaere et al. 1995b).
A chimeric gene containing the CaMV 355 promoter and
the 5' end of the poplar OMT gene in antisense orientation
was introduced into tobacco by Dwivedi et al. {1994). In
some transgenic tobacco plants, the level of OMT activity
was decreased and modification of the lignin composition
was observed. This demonstrates that homology between
the tobacco and poplar OMT genes is sufficient for a functional antisense strategy. Another study reported under
expression of OMT in transgenic poplars expressing a poplar antisense RNA (van Doorsselaere et al. 1995b ). OMT
activity was reduced up to 95 percent and critical modification of the lignin composition was observed. Reducing
the activity of enzymes involved in lignin monomer biosynthesis is potentially important for the paper pulp industry and can be effectively tested in woody plants.
Detailed information on this subject is included in the chapter by Boerjan et al. in this volume.
Summary
Poplar is considered a model plant for gene expression
studies in woody angiosperm species. The advantages of
the poplar model system are the availability of transformation procedures and its small genome size, which allows for relatively easy gene isolation. High levels of
homology and conservation of the regulatory systems have
been observed between poplar (a dicot) genes and other
67
Section II Transformation and Foreign Gene Expression
dicots. Poplar promoters and genes potentially can be studied in other model plant species such as tobacco. However, for specific metabolic pathways, such as lignification,
more representative information will result if gene expression is tested in poplar. The ability to transform many poplar genotypes affords diverse opportunities to study
expression of genes from other woody species, including
conifers. In the future, the development of such studies
could provide critical information on gene expression in
woody plants.
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