(PAL), chalcone synthetase (CHS), the flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase
(DFR).
The first enzyme, PAL, is the only one not directly associated with the flavonoid pathway. It is a key
enzyme catalyzing the first step from phenylalanine to hydroxycinnamic acid, the deamination of pheny-
lalanine to cinnamic acid. Hydroxycinnamic acid is required for flavonoid biosynthesis. Two cDNA
clones were isolated for Bromheadia finlaysonianaPAL but only one was sequenced to completion [21].
In several other species, PAL is encoded by a small family of genes, although loblolly pine contains only
a single PAL gene [88,89].
The second enzyme, CHS, catalyzes the formation of chalcones as indicated earlier. Three cDNA
clones were isolated for B. finlaysonianaCHS [12], and evidence was obtained for multiple CHS genes.
Further evidence indicated the presence of transcripts in all floral organs and plant parts. However, defini-
tive experiments to differentiate the transcripts corresponding to individual cDNA clones have not been
reported. The analysis of CHS genes in different plant species has shown variability in copy number rang-
ing from 1 to 10 copies per genome [90–94]. Individual members of CHS multigene families can be dif-
ferently regulated. In the case of petunia, it is known that only one of the CHS genes is strongly expressed
in petal tissue [95]. A cDNA clone was also obtained for Phalaenopsissp. ‘True lady’ CHS [11]. The de-
duced amino acid sequence of the latter CHS is 60–64% identical to that of B. finlaysonianaCHS. We
did the sequence comparison using an ALIGN program [36]. Based on results of Southern hybridization,
thePhalaenopsisCHS gene was reported to exist in multiple copies.
The third enzyme, F3H, is responsible for the conversion of flavanones to dihydroflavonols, the rel-
ative expression of its gene affecting the production of different anthocyanins. A cDNA clone was re-
ported for B. finlaysonianaF3H [18].
The last enzyme, DFR, catalyzes the first committed step to anthocyanin biosynthesis by converting
dihydroflavonols into leucoanthocyanidins, the immediate precursors for anthocyanins. One cDNA clone
each was obtained for B. finlaysonianaDFR [96] and Cymbidium hybridaDFR [14], and the deduced
amino acid sequence of the former DFR is 85% homologous to that of the latter DFR. Evidence was also
obtained for a single copy of the DFR gene in the petal tissue of either orchid.
Although the number of molecular studies of anthocyanin biosynthesis in orchid is limited, the
cloned gene for C. hybridaDFR has already been used in a transgenic approach to understand the molec-
ular factors affecting the color range of some species of Cymbidiumflowers that conspicuously lack or-
ange-colored flowers [97]. Pelargonidin is the anthocyanin that normally leads to orange pigmentation.
When the C. hybridaDFR gene was transformed into a DFRpetunia line, the C. hybridaDFR was found
not to efficiently reduce dihydrokaempferol to leucopelargonidin, a substrate required for pelargonidin
production. These results suggested that the lack of orange-colored Cymbidiumflowers is due to the loss
of preference for dihydrokaempferol as a substrate, a phenomenon also observed in the DFR preparations
fromPetunia, Lycoperscion, and Nicotianaplants [98].
In addition to the preceding cloned genes, there are orchid genes isolated from nonflower tissue, and
some of these are relevant to the understanding of floral coloration. The availability of these genes will
help in the isolation of their counterparts in petal tissue. The enzymes for which cDNA clones have been
isolated from nonflower tissue include 4-coumarate:coenzyme A ligase [13], caffeic acid O-methyltrans-
ferases [19,20], S-adenosylhomocysteine hydrolase [10], bibenzyl synthase [9,10], and vacuolar H-ATP
synthase 16-kDa proteolipid subunit [99]. Except for the last enzyme, the rest are involved in pathways
that interact with that of the flavonoid. By virtue of their catalytic activities, these enzymes affect either
directly or indirectly the biosynthesis of flavonoids. The role of vacuolar H-ATP synthase in floral col-
oration has not been investigated before. This enzyme pumps protons across vacuolar membrane at the
expense of ATP and brings about a pH change in the vacuole or cytosol. It is also known that the antho-
cyanins are stored in the vacuole, whose pH has a marked effect on the anthocyanin secondary structures
and thus on the resultant floral coloration. Taken together, it is hard not to imagine that the vacuolar H-
ATP synthase can influence floral coloration.
V. CONCLUSION
This chapter focuses on the orchid flower genes and cites many more recent references than an earlier sur-
vey concluded in 1994 [100]. Although our knowledge of each of the research areas reviewed is still rudi-
mentary, the cloning of these genes will pave the way to address some of the questions raised in the in-
GENES ASSOCIATED WITH ORCHID FLOWER 557