III. POLLINATION OF FLOWERS
The study of flower pollination has long attracted interest because of the developmental changes that take
place following the pollination event and the fact that these changes contribute to successful reproduc-
tion. The major physiological and morphological changes in orchid flowers following pollination have
been described in great detail [39]. Of these changes, ovule differentiation, perianth senescence, and ethy-
lene biosynthesis have been the central thrust of molecular studies. The model orchid system Phalaenop-
sishas been the source of many of the genes involved in these changes to date.
A. Ovule Development
Orchid is unusual in the sense that in many of its species, ovule development is induced by pollination.
Following induction, the development of the megasporocyte and megagametophyte is not much different
from that in many other plant species, and the subsequent maturation of the embryo sac conforms to the
Polygonumtype [40]. Characterization of gene expression associated with ovule differentiation in Pha-
laenopsissp. SM9108 has been aided by defining the timing and hormonal stimuli of each developmen-
tal transition stage [41]. The isolation of genes O39, O40, O108, O126, O141[15], and O138[42] from
Phalaenopsisis further facilitated by establishing stage-specific cDNA libraries derived from the arches-
porial cell, megaspore mother cell, and mature embryo sac and screening for cDNA clones differentially
expressed at each of these stages.
O39encodes a 84-kDa putative homeobox transcription factor and is expressed in the ovule from
formation of the ovule primordium at early stages through to various stages of ovule tissue differentia-
tion. On the basis of its pattern of expression and its strong similarity at the N-terminal to the homeobox
DNA binding motif of transcription factors, O39is thought to be involved in the initiation of ovule de-
velopment. Alternatively, O39may be induced in response to the commitment to ovule differentiation,
and it may in turn regulate a subset of genes involved in the developmental pathway to ensure appropri-
ate differentiation. Using O39as a probe to screen an Arabidopsisfloral bud cDNA library resulted in
identification of the ArabidopsishomologueATML1[43].ATML1is expressed specifically in the L1
layer of the meristem from the earliest stages of meristem patterning and throughout shoot development,
suggesting that ATML1may be involved in setting up morphogenetic boundaries of positional informa-
tion necessary for controlling cell specification and pattern formation. ATML1is also thought to provide
an early molecular marker for the establishment of both apical-based radical patterns during plant em-
bryogenesis. As with other homeobox protein families, PhalaenopsisO39 has little sequence similarity
to other homeobox protein families outside the homeobox region. A notable exception is Arabidopsis
GLABRA2 (GL2), which regulates trichome differentiation, and ATML1. O39, together with the Ara-
bidopsisGLABRA2 (GL2) and ATML1, is proposed to define a new class of plant homeodomain-con-
taining proteins designated HD-GL2.
O40encodes a cytochrome P450 monooxygenase of predicted molecular mass 48 kDa. The large su-
perfamily of cytochrome P450 monooxygenases are membrane-bound enzymes that catalyze the oxida-
tion of diverse and often overlapping substrates of both endogenous and xenobiotic origin in bacteria,
fungi, plants, and animals [44,45]. O40 is the first orchid cytochrome P450 identified, and by convention
it represents the second of the CYP78 gene family and consequently is named CYP78A2 by the Cy-
tochrome P450 Gene Nomenclature Committee. In situ hybridization has shown that O40 messenger
RNA (mRNA) is not specific to ovules but is found exclusively in the pollen tubes intertwined with the
ovules during early ovule development. The function of O40 is as yet unclear, although it is thought to
play a role in the biosynthesis of a hormone involved in intercellular communication, similar to its ho-
mologue in the animal systems.
In contrast to O39andO40, O108, O126, and O141are selectively expressed in maturing ovules.
The 15-kDa putative protein encoded by O108contains a consensus ATP/GTP binding site at the C-ter-
minal.O108is expressed in actively dividing cells of the ovule, exclusively in the outer layer of the outer
integument and in the female gametophyte of mature ovules. When O108was initially cloned, it showed
no obvious similarity to any other gene with a known function in the database. O108was then thought to
be important for successful seed production, perhaps by facilitating signal transduction between the ovule
and pollen tube. Based on the sequence of O108, the ArabidopsishomologueAtskp1has been identified
[46].Atskp1is expressed in all actively dividing cells of the plant body, particularly in plant meristem.
552 NEO AND HO