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.

Arabidopsis chotto1 (cho1) mutants show resistance to (-)-R-ABA, an ABA analog, during germination and seedling growth. Here, we report cloning and characterization of the CHO1 gene. cho1 mutants showed only subtle resistance to (+)-S-ABA during germination. The cho1 mutation acts as a strong enhancer of the abi5 mutant, whereas the cho1 abi4 double mutant showed ABA resistance similar to the abi4 single mutant. This suggests that CHO1 and ABI4, but not ABI5, act in the same genetic pathway. Map-based cloning revealed that the CHO1 gene encodes a putative transcription factor containing double AP2 domains. The CHO1 gene was expressed predominantly in seed, with the strongest expression in imbibed seed. Induction of CHO1 expression was observed 4 h after seed imbibition and reached a maximum level at 24 h. Induction of CHO1 expression did not occur in the abi4 mutants, indicating that this is an ABI4-dependent process. Microarray experiments showed that a large number of genes involved in primary metabolism and the stress response were up-regulated in the cho1 mutant. Growth of abi4 and cho1 mutant seedlings was resistant to high concentrations of glucose. In addition, growth of cho1 mutant seedlings was partially resistant to excess nitrate (50 mM), as evident from their expanded green cotyledons. However, their growth was normal under moderate nitrate concentrations (< 10 mM). This nitrate response was specific to the cho1 mutants and was not observed in the abi4 mutants. Taken together, our results indicate that CHO1 regulates nutritional responses downstream of ABI4 during germination and seedling growth.

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I–III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87% of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method.