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Plant Physiol, December 2001, Vol. 127, pp. 1390-1393
SCIENTIFIC CORRESPONDENCE
Molecular Genetics of Reproductive Biology in Orchids
Hao
Yu and
Chong Jin
Goh*
Plant Growth and Development Laboratory, Department of Biological
Sciences, Faculty of Science, National University of Singapore, 10 Kent
Ridge Crescent, Singapore 119260, Republic of Singapore
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INTRODUCTION |
Orchids are members of the family
Orchidaceae, one of the largest families of flowering plants.
There are an estimated 20,000 to 25,000 orchid species, which occupy
wide ranges of ecological habitats and exhibit highly specialized
morphological, structural, and physiological characteristics (Dressler,
1990 ). In particular, the most spectacular evolution is shown in
reproductive biology. The production of column (a fused structure of
stamens and styles) to facilitate pollination is well documented and
the co-evolution of orchid flowers and pollinators is well known (van
der Pijl and Dodson, 1969). Even more significant but less well-known
aspects are the early development and maturation of the pollen grains (packaged as pollinia pollen grains bound together by viscin threads in masses for effective pollination), the post-pollination development and maturation of ovules, the synchronized timing of micro- and mega-gametogenesis for effective fertilization along the whole length
of placenta, and the release of tens of thousands or millions of
immature embryos (globular stage) in mature capsules (Raghavan and Goh,
1994). Without doubt, these various strategies, unique to orchids,
contribute to the success of orchid family.
During the past decade, intensive molecular studies with the model
plant Arabidopsis and with rice (Oryza sativa) have
elucidated many gene regulation processes during development,
particularly on reproductive biology of flowering. These studies would
be further enhanced greatly with the complete sequencing of Arabidopsis
and rice genomes and the numerous expressed sequence tag sequencing projects in a wide range of plants. In contrast, few molecular genetic
investigations have so far been undertaken on floral transition and
subsequent reproductive growth in orchids. This was, in some way,
limited by the rather extended and complicated flowering process. The
inefficient orchid transformation system also hampered the
investigation of gene function and regulation in vivo. However, recent
successes in in vitro thin-section techniques for micropropagation and
flowering of orchids have not only shortened the orchid juvenile phase
from several years to only a few months but also provided more obvious
"landmark" events during development (Lakshmanan et al., 1995; Goh,
1996). Also, the improved orchid transformation system (Chia et al.,
1994; Yang et al., 1999; Yu et al., 2001) would certainly facilitate
studies on gene function. Progress in molecular genetics of orchids can
be expected to accelerate in the near future. The unique differences in
reproductive biology in orchids as compared with the normal development
in Arabidopsis offer distinct advantages to study gene function and
evolution in prepollination and post-pollination development.
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PREPOLLINATION: FLOWER DEVELOPMENT |
Our understanding of prepollination floral development in orchids
at the molecular level is only at the initial stage. In some plant
species, rapid progress has been made in elucidating the molecular and
genetic mechanisms involved in the floral transition and subsequent
flower development. In particular, a large number of MADS-box genes,
which encode transcription factors containing a highly conserved
DNA-binding domain, have been identified to function in subtle
regulatory processes of flower development in different species (Shore
and Sharrocks, 1995; Riechmann and Meyerowitz, 1997; Kyozuka et al.,
2000). Using the in vitro flowering system of Dendrobium
spp. orchids, the profile of gene expression during the transition to
flowering has been examined by mRNA differential display method (Yu and
Goh, 2000a). The results showed that genes involved in transcriptional
regulation, cell division, and several other metabolic
events are closely associated with the process of floral transition
in orchids. Furthermore, four orchid members of the APETALA1/AGL9
subfamily of the MADS-box gene have been identified in
Aranda cv Deborah and Dendrobium cv
Madame Thong-In (Lu et al., 1993; Yu and Goh, 2000b). Study of the
expression patterns of these genes indicated their important roles in
the regulation of floral transition and organ identity (Yu and Goh, 2000b).
It is noteworthy that during flower development, interaction may occur
between MADS-box and class 1 knox genes, another
developmentally important class of transcription factors (Yu et al.,
2000). This interesting hypothesis needs further investigation in
orchids and other plant species. In cv Madame Thong-In, down-regulation of the expression of DOH1 gene, a class 1 knox
gene, causes multiple shoot apical meristem formation and early
flowering, which is coupled with the early onset expression
of DOMADS1, an orchid MADS-box gene involved in the floral
transition (Yu et al., 2000). In a wild-type orchid plant, the onset of
DOMADS1 expression in the shoot apical meristem during
floral transition is accompanied by a marked reduction of
DOH1 transcripts, and both kinds of transcripts are later
located at the same region in the inflorescence meristem and the
developing floral primordia (Yu et al., 2000). Therefore, one can
reasonably envisage a possible relationship between these two different
types of transcription factors during the flowering process.
The extensive molecular and genetic studies of Arabidopsis and rice
genomes as well as physiological responses have described detailed
regulatory networks of flower development including several pathways
leading to the onset of flowering, and the initiation and formation of
floral meristem and organs. These studies will undoubtedly play
directional roles in future elucidation of basic mechanisms of flower
development in orchids. On the other hand, unique and different
developmental programs may be present in orchids due to the highly
evolved floral structures, which are being investigated to contribute
to our understanding of the molecular events regulating the general
floral development in flowering plants. For example, in contrast to the
homologs in Arabidopsis, the isolated orchid MADS-box genes,
DOMADS2 and DOMADS3, have shown novel expression
patterns in the shoot apical meristem during floral transition (Yu and
Goh, 2000b).
It should be noted that, although floral organ identity (ABC)
genes have been well studied in Arabidopsis and rice, no such homologs have been isolated from orchid. The development of column, which involves whorl 3 and whorl 4, would be one of the most
interesting subjects to elucidate the evolution and interaction of
B and C genes. Indeed, clarification of the early
development of pollinia with respect to other floral organs would
provide insights on regulation of microspore genesis.
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PREPOLLINATION: PIGMENTATION |
Compared with Arabidopsis and rice, orchid is ideal for the study
of floral coloration because of its enormous variation in color. Some
investigations have shed light on the clarification of genes affecting
orchid flower pigmentation, most of which encode enzymes relevant to
the flavonoid pathway (Liew et al., 1998; Johnson et al., 1999). These
genes include dihydroflavonol 4-reductase (DFR), chalcone synthetase, flavanone
3-hydroxylase, and Phe ammonia-lyase. However,
molecular and genetic studies of these genes are too limited to figure
out the fundamental factors regulating the floral coloration of
orchids, although the transgenic approach has been adopted for the
study of Cymbidium hybrida DFR in a heterologous petunia (Petunia hybrida) system (Johnson et al.,
1999). Orchid flowers are striking for their specific patterns of
colors in sepals, petals, and the modified dorsal petals (lips). These
may be discrete spots, streaks, or blotches rather than flushes or shades of different intensity. The patterns on the lips of some species
are even more strikingly contrasting to serve as the landing platform
for insect pollinators. It follows that the regulation of pigmentation
is refined to specific cells of the different floral organs, and the
expression of genes involved in flavonoid synthesis may be just the
initial steps in the complex regulation of pigmentation. Indeed,
DFR was shown to be expressed in the white petal tissues of
Bromheadia finlaysoniana (Liew et al., 1998). To understand
the development of color patterns in flowers, it will be also important
to identify additional regulatory genes in the flavonoid pathway.
Further investigations of their regulatory mechanisms and pathways will
provide strategies toward genetic engineering of color in orchid
flower, which is now possible with efficient and reliable
transformation systems (Chia et al., 1994; Yang et al., 1999; Yu et
al., 2001).
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POST-POLLINATION: OVULE DEVELOPMENT |
In most of flowering plants, the ovules are mature, and the egg
cells are ready to be fertilized at anthesis. In contrast, ovule
development is triggered by pollination in orchids. This makes orchids
attractive systems for the investigation of ovule initiation and
subsequent development. Molecular aspects of ovule development have
been investigated in Phalaenopsis spp. orchids (O'Neill et
al., 1993; Zhang and O'Neill, 1993; Nadeau et al., 1996). These
studies led to the isolation and characterization of a series of orchid
genes associated with ovule differentiation, which provided insights
into the function of their homologs in Arabidopsis development (Lu et
al., 1996; Porat et al., 1998). For example, the Phalaenopsis
O39 gene is a member of a new class of plant homeobox
transcription factors designated HD-GL2 (Lu et al., 1996; Nadeau et
al., 1996). It is expressed in the ovule from primordium formation at
early stages to various late stages of ovule differentiation,
suggesting its possible role as an important regulator involved in the
ovule tissue initiation and development. Subsequently, the Arabidopsis
homolog of O39, ATML1, has been identified in an
Arabidopsis floral bud cDNA library using the O39 probe (Lu
et al., 1996). The ATML1 transcript is located in the L1
layer of the meristem during embryonic pattern formation and throughout
shoot development, which indicates that ATML1 may participate in the meristem patterning from the earliest stages of
embryogenesis to the later stages of shoot development.
The critical role of ethylene in ovary maturation and ovule
differentiation in orchids has been extensively investigated by both
physiological and molecular methods (O'Neill et al., 1993; Nadeau et
al., 1996). Most of these studies were focused on two components,
1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase,
both of which are key enzymes in the ethylene biosynthetic pathway.
Although evidence has been presented for the coordinated regulation of
ACC synthase and ACC oxidase gene expression in the ovary (O'Neill et
al., 1993), the molecular events triggered by pollen-pistil
interactions leading to these gene expressions are not well understood.
Furthermore, the signaling pathway following ethylene perception is
still not clarified during post-pollination gynoecium changes in orchids.
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POST-POLLINATION: PERIANTH SENESCENCE/DEVELOPMENT |
Perianth senescence induced by pollination in orchids is a typical
post-pollination symptom with rapid sepal and petal wilting and pigment
loss. Successful pollination signals the completion of a job well done
by the perianth display, and the resources are then redirected for
ovule development and subsequent embryogenesis after fertilization. The
physiological and molecular mechanisms of pollination-induced
senescence have been studied in several orchid species, such as
Phalaenopsis and Dendrobium, especially in terms
of both ethylene sensitivity and production. The enhanced sensitivity
to ethylene following pollination is the initial event triggering an
increase in ethylene production and the consequent physiological
changes of flower (Porat et al., 1995). Although molecular evidence is
hitherto far from resolving pollen-derived signals, the identification
of some putative sensitivity factors such as GTP-binding protein (Porat
et al., 1994), short-chain saturated fatty acids (Halevy et al., 1996),
and auxin (Zhang and O'Neill, 1993) has shed light on the elucidation
of the mechanisms under the regulation of ethylene sensitivity.
Increase in ethylene production is another early event induced by
pollination, which occurs after a heightened sensitivity to ethylene in
orchid flowers. In Phalaenopsis spp., despite the production
of abundant ethylene in the perianth up to 72 h after pollination,
the accumulation of ACC synthase is not detectable in this tissue
(O'Neill et al., 1993). However, the ACC oxidase expression is
up-regulated in the petals and sepals about 48 h after pollination
in parallel with the onset of perianth senescence. It is generally
accepted that both ACC synthase and ACC oxidase, positively regulated
by ethylene, function in a feedback loop, leading to the increased
ethylene production (O'Neill et al., 1993).
At the other extreme, in some Phalaenopsis species
(subdivision stauroglottis), the sepals and petals turn green and
photosynthetic following successful pollination. These organs become
leaf-like and provide photosynthates for the developing ovules/ovary
and the embryos subsequent to fertilization over an extended period of
many months until the capsule is mature. The molecular genetics for
this transformation of the perianth from an energy sink to an energy
source during post-pollination development is another marvelous
opportunity to study the interaction (suppression?) of ABC genes.
The reproductive biology in orchids is still lagging behind in terms of
molecular genetics. Information on the genomes of Arabidopsis and rice,
together with large collections of expressed sequence tag, is providing
new strategies for addressing the universal knowledge of plant systems
in a more integrated perspective. Application of this knowledge through
the common language of nucleotide sequences will allow the assignation
of potential functions to the corresponding genes in orchids, and thus
partially direct further characterization of these candidates involved
in the important developmental processes. On the other hand, gene
cloning in orchids, as compared with similar work in other plant
species, will also contribute to studies of the functions of specific
genes, which are yet to be or have not been completely determined in
Arabidopsis or rice. As another kind of flowering plants, orchids
demonstrate some special characteristics and offer unique advantages in
the study of certain important developmental programs, such as floral
coloration, ovule development, and perianth senescence/development.
Future concentration of efforts on one orchid species, which is easily
transformable with relatively detailed genetic map, will promise an
exciting future for orchidology with a fusion of classical plant
physiology and modern molecular genetics.
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FOOTNOTES |
Received July 31, 2001; accepted August 20, 2001.
*
Corresponding author; e-mail dbsgohcj{at}nus.edu.sg; fax
65-779-5671.
www.plantphysiol.org/cgi/doi/10.1104/pp.010676.
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© 2001 American Society of Plant Physiologists
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TOP
INTRODUCTION
PREPOLLINATION: FLOWER...