The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8 0 -hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8 0 -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 0 -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.

Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 89-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 89-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 89-hydroxylation of (1)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (1)-ABA to yield phaseic acid, the isomerized form of 89-hydroxy-ABA. The microsomes from the insect cells exhibited very strong activity of 89-hydroxylation of (1)-ABA (K m ¼ 1.3 mM and k cat ¼ 15 min ÿ1 ). The solubilized CYP707A3 protein bound (1)-ABA with the binding constant K s ¼ 3.5 mM, but did not bind (ÿ)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 89-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 89-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 89-hydroxylation of (1)-ABA.

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 latematuration. 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 midmaturation. 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.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

No abstract

Plants in the Nicotiana genus produce nicotine and related pyridine alkaloids as a part of their chemical defense against insect herbivores. These alkaloids are formed by condensation of a derivative of nicotinic acid, but the enzyme(s) involved in the final condensation step remains elusive. In Nicotiana tabacum, an orphan reductase A622 and its close homolog A622L are coordinately expressed in the root, upregulated by methyl jasmonate treatment, and controlled by the NIC regulatory loci specific to the biosynthesis of tobacco alkaloids. Conditional suppression of A622 and A622L by RNA interference inhibited cell growth, severely decreased the formation of all tobacco alkaloids, and concomitantly induced an accumulation of nicotinic acid beta-N-glucoside, a probable detoxification metabolite of nicotinic acid, in both hairy roots and methyl jasmonate-elicited cultured cells of tobacco. N-methylpyrrolinium cation, a precursor of the pyrrolidine moiety of nicotine, also accumulated in the A622(L)-knockdown hairy roots. We propose that the tobacco A622-like reductases of the PIP family are involved in either the formation of a nicotinic acid-derived precursor or the final condensation reaction of tobacco alkaloids.

No abstract

No abstract