mononucleotide (FMN) binding site [66]. The genomic structure of PACO1 encompasses approximately
14 kb and is divided into seven exons by six introns [67]. Southern blot analysis indicates that the gene
occurs in one copy or a low number of copies per haploid genome, and Northern analysis shows two
species of alternatively spliced mRNA. Understanding the complexity of the PACO1 gene structure and
its regulation should further elucidate the role of -oxidation in flower senescence.
A major event occurring after pollination is an increase in ethylene production, which is generally
accepted to be a primary signal for pollination-induced senescence [40,53]. After the physical event of
pollination, ethylene evolution occurs in the stigma, possibly due to triggering by pollen-borne auxin and
other factors, followed by endogenous synthesis of ACC. The details of ethylene biosynthesis and the in-
terorgan regulation are discussed in the next section.
C. Ethylene Biosynthesis in Pollinated Flowers
The ethylene biosynthetic pathway involves the conversion of S-adenosylmethionine to ACC by ACC
synthase. The ethylene precursor ACC is then converted to ethylene by ACC oxidase [68]. ACC synthase
and ACC oxidase are therefore key enzymes in ethylene biosynthesis, although ACC synthase is gener-
ally considered to be rate limiting. Both ACC synthase and ACC oxidase genes are encoded by multigene
families and have been well characterized in many plant species [69,70]. ACC synthase genes exhibit dif-
ferential tissue specificity and are regulated by environmental or hormonal stimuli [68,70]. ACC oxidase
genes are considered in most cases to be mostly constitutive [71]. However, there is compelling evidence
that ACC oxidase genes are differentially expressed and are highly regulated in floral organs [69,72,73].
In addition, both ACC synthase and ACC oxidase contribute to a positive feedback loop wherein ethy-
lene treatment leads to increased ethylene production [74].
In orchids, three full-length ACC synthase genes, Ds-ACS1, Ds-ACS2[3] and Pt-ACS1[6], have
been identified in Phalaenopsis. Their predicted protein sequences comprise 425, 444, and 445 amino
acids, respectively. In addition, an ACC synthase gene DC-ACS, which is predicted to encode a 435-
amino-acid protein, has been isolated from Dendrobium crumenatum[7].Ds-ACS1andDs-ACS2ap-
pear to be different transcripts of the same gene, and their predicted protein sequences show 97% sim-
ilarity (Figure 2). In addition, Ds-ACS1is reported to be homologous to Phal-ACS1[75]. Pt-ACS1
shares 68%, 67%, and 74% similarity with Ds-ACS1, Ds-ACS2, and DC-ACS, respectively (Figure 3).
DC-ACS is highly similar to Ds-ACS1 (84% similarity) and Ds-ACS2 (85% similarity). The partial se-
quences of two other distinct PhalenopsisACC synthase genes, Phal-ACS2andPhal-ACS3, have been
cloned by reverse transcription–polymerase chain reaction (RT-PCR) [75], indicating that Phalaenop-
sisACC synthase consists of at least three gene members. These ACC synthase genes show differen-
tial expression in floral organs in response to pollination and various chemical stimuli. Phal-ACS2and
Phal-ACS3genes are expressed within 1–2 hr after pollination in the stigma and ovary, respectively,
whereas the expression of Phal-ACS1is detected only in the stigma 6 hr after pollination. Furthermore,
Phal-ACS2andPhal-ACS3are induced in response to primary pollination signals such as auxin,
whereas Ds-ACS1 is induced in response to secondary pollination signals such as ACC. This suggests
that orchid flowers have at least two different types of ACC synthase genes, one responding to the pri-
mary pollination signal and another amplifying the primary signal by triggering and/or sustaining au-
tocatalytic ethylene production. The auxin-induced Phal-ACS3 mRNA accumulation in the ovary is
severalfold less than that induced by pollination, indicating that an unknown pollination factor may
have a synergistic effect with auxin. The differential expression of Phal-ACS1andPhal-ACS2in the
stigma of pollinated orchids suggests that their combined expression may be responsible for the de novo
synthesis of ACC required for sustained ethylene production in the stigma. Similar regulation of ACC
synthase in carnation has been shown [76].
For ACC oxidase, two genes, OAO1[3,73] and D-ACO2[4], have been isolated from Phalaenopsis
and one gene, DCACO, has been identified in Dendrobium[5]. Their predicted protein sequences consist
of 317, 318, and 325 amino acids, respectively. Sequence comparison indicates that these ACC oxidase
proteins are highly similar to each other. D-ACO2 shares 94% similarity with OAO1, and DCACO shares
85% similarity with both OAO1 and D-ACO2 (Figure 3). The regulation of ACC oxidase activity and
mRNA expression within the floral organs in Phalaenopsisorchid after pollination have been examined.
Rapid induction of ACC oxidase activity and high accumulation of the OAO1 mRNA is observed fol-
lowing pollination. Furthermore, ACC oxidase gene expression is regulated by ethylene.
554 NEO AND HO