Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C 15 intermediate xanthoxin and C 25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9-cisneoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located. A bscisic acid (ABA) is one of the five ''classical'' plant hormones (1). It is ubiquitous in higher plants and algae and is also produced by several fungi (2). In higher plants, ABA plays important roles in embryo development and seed dormancy. ABA is also involved in adaptation of plants to various stresses (drought, salinity, cold). When ABA is applied to plants, it causes rapid stomatal closure and thus reduces water loss via transpiration. Furthermore, water stress causes a rapid increase in the ABA content of plants. Evidence has accumulated that this increase in ABA is attributable to de novo synthesis (3). The increase in ABA biosynthesis in water-stressed plants can be prevented by application of actinomycin D, cordycepin, or cycloheximide (4, 5), indicating that nuclear gene transcription and protein synthesis in the cytosol are required before an increase in ABA synthesis can occur.To determine which step(s) in ABA biosynthesis is (are) stimulated by water stress, it was essential to first establish the ABA biosynthetic pathway. ABA is a sesquiterpenoid (C 15 ), and two pathways for its biosynthesis have been proposed: (i) The direct pathway from isopentenyl pyrophosphate (C 5 ) via farnesyl pyrophosphate (C 15 ) to ABA. Current evidence indicates that this pathway operates in fungi (2). (ii) The indirect pathway in which ABA is a cleavage product of carotenoids (C 40 ...

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9 -10 and 9-10 positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C 14 dialdehyde and two C 13 products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.

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Enzymes that are able to oxidatively cleave carotenoids at specific positions have been identified in animals and plants. The first such enzyme to be identified was a nine-cis-epoxy carotenoid dioxygenase from maize, which catalyzes the rate-limiting step of abscisic acid biosynthesis. Similar enzymes are necessary for the synthesis of vitamin A in animals and other carotenoidderived molecules in plants. In the model plant, Arabidopsis, there are nine hypothetical proteins that share some degree of sequence similarity to the nine-cis-epoxy carotenoid dioxygenases. Five of these proteins appear to be involved in abscisic acid biosynthesis. The remaining four proteins are expected to catalyze other carotenoid cleavage reactions and have been named carotenoid cleavage dioxygenases (CCDs). The hypothetical proteins, AtCCD7 and AtCCD8, are the most disparate members of this protein family in Arabidopsis. The max3 and max4 mutants in Arabidopsis result from lesions in AtCCD7 and AtCCD8. Both mutants display a dramatic increase in lateral branching and are believed to be impaired in the synthesis of an unidentified compound that inhibits axillary meristem development. To determine the biochemical function of AtCCD7, the protein was expressed in carotenoid-accumulating strains of Escherichia coli. The activity of AtCCD7 was also tested in vitro with several of the most common plant carotenoids. It was shown that the recombinant AtCCD7 protein catalyzes a specific 9 -10 cleavage of ␤-carotene to produce the 10-apo-␤-carotenal (C 27 ) and ␤-ionone (C 13 ). When AtCCD7 and AtCCD8 were co-expressed in a ␤-carotene-producing strain of E. coli, the 13-apo-␤-carotenone (C 18 ) was produced. The C 18 product appears to result from a secondary cleavage of the AtCCD7-derived C 27 product. The sequential cleavages of ␤-carotene by AtCCD7 and AtCCD8 are likely the initial steps in the synthesis of a carotenoid-derived signaling molecule that is necessary for the regulation lateral branching.

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.Abscisic acid (ABA) is necessary for seed maturation and dormancy and adaptation to a variety of environmental stresses. The role of ABA in these processes is mediated by changes in gene expression and stomatal closure. Mutants impaired in ABA biosynthesis display precocious germination and have a wilty phenotype (Zeevaart, 1999). Application of ABA results in delayed germination and increased tolerance to a variety of stresses.It is now well established that ABA in higher plants is derived from C 40 -carotenoids (Cutler and Krochko, 1999;Liotenberg et al., 1999;Taylor et al., 2000). Epoxidation of the C 40 -carotenoid, zeaxanthin, gives rise to all-trans-violaxanthin, one of the xanthophylls in higher plants. This reaction is catalyzed by zeaxanthin epoxidase (ZEP). The ZEP gene was isolated from the aba2 mutant of wild tobacco (Nicotiana plumbaginifolia) and the aba1 mutant of Arabidopsis (Marin et al., 1996). The xanthophylls also include three other isomers, 9-cis-violaxanthin, all-transneoxanthin, and 9-cis-neoxanthin. Genes encoding neoxanthin synthase have been cloned from potato (Solanum tuberosum; Al-Babili et al., 2000) and tomato (Lycopersicon esculentum; Bouvier et al., 2000).The first C 15 precursor of ABA is xanthoxin, which is a cleavage product of C 40 -epoxy-carotenoids. Current evidence indicates that the key regulatory step in ABA biosynthesis is the oxidative cleavage reaction . The gene encoding the cleavage enzyme was first cloned from the ABA-deficient muta...