The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′‐hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′‐hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1 – CYP707A4 , encode ABA 8′‐hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six‐fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

Endogenous abscisic acid (ABA) levels are regulated by both biosynthesis and catabolism of the hormone. ABA 8′-hydroxylase is considered to be the key catabolic enzyme in many physiological processes. We have previously identified that four members of the Arabidopsis (Arabidopsis thaliana) CYP707A gene family (CYP707A1 to CYP707A4) encode ABA 8′-hydroxylases, and that the cyp707a2 mutants showed an increase in ABA levels in dry and imbibed seeds. In this study, we showed that the cyp707a1 mutant accumulated ABA to higher levels in dry seeds than the cyp707a2 mutant. Expression analysis showed that the CYP707A1 was expressed predominantly during mid-maturation and was down-regulated during late-maturation. Concomitantly, the CYP707A2 transcript levels increased from late-maturation to mature dry seed. Phenotypic analysis of single and double cyp707a mutants indicates that the CYP707A1 is important for reducing ABA levels during mid-maturation. On the other hand, CYP707A2 is responsible for the regulation of ABA levels from late-maturation to germination. Moreover, CYP707A1 and CYP707A3 were also shown to be involved in postgermination growth. Spatial expression analysis suggests that CYP707A1 was expressed predominantly in embryo during mid-maturation, whereas CYP707A2 expression was detected in both embryo and endosperm from late-maturation to germination. Our results demonstrate that each CYP707A gene plays a distinct role during seed development and postgermination growth.

Summary: Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8′‐hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T‐DNA insertional cyp707a3‐1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3‐1 mutant accumulated a higher amount of stress‐induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA‐inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.

β‐amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)‐2,3‐oxidosqualene. The enzyme, β‐amyrin synthase, catalyzing the cyclization of oxidosqualene into β‐amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology‐based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane‐type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3′‐RACE and 5′‐RACE was carried out to obtain the whole sequences. The two clones exhibited 60 % amino acid identity to each other. A full‐length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSC_PNY . β‐amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSC_PNY contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSC_PNX, was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8′-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8′-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1–green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1–2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.