SummaryDual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is known to be induced by environmental stresses and regulated developmentally. We used a negative-selection approach to isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized three recessive mutants, aar1-1, aar1-2, and aar2-1, which belong to two complementation groups. In addition to their defects during seed germination, mutations in the AAR1 and AAR2 genes also affected anoxic and hypoxic induction of ADH and other glycolytic genes in mature plants. The aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1-1and aar1-2 exhibited different phenotypes under cold and osmotic stresses. Based on our results we propose that these mutants are defective in a late step of the signaling pathways that lead to increased expression of the ADH gene and glycolytic genes.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.

We have characterized the effects of different light spedra on expression of the nuclear genes (GapA and GapS) encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis fhaliana. Steady-state mRNA levels for both genes in etiolated seedlings increased after a short exposure to red or blue light. However, these increases could not be reversed by immediate farred light following the initial light treatment. In mature plants, a short light pulse, regardless of its spedrum, had no apparent effect on GapA or GapS mRNA levels in dark-adapted plants. In contrast, continuous exposure to red, blue, or white light resulted in increases of GapA and GapS mRNA levels, with blue and white light being far more efficient than red light. Similarly, continuous exposure of etiolated seedlings to red, blue, or white light also resulted in increased CapA and CapB mRNA levels. In addition, we show that illumination of red light-saturated Arabidopsis plants with continuous blue light results in further increases of GapA and GapS mRNA levels. Based on these results, we conclude that both blue light photoreceptor-and phytochrome-mediated pathways are involved in light regulation of GapA and GapS genes in Arabidopsis, with blue light acting as the dominant regulator.

The mechanisms by which plants detect water deficit and transduce that signal into adaptive responses is unknown. In maize (Zea mays L.) seedlings, primary roots adapt to low water potentials such that substantial rates of elongation continue when shoot growth is completely inhibited. In this study, in-gel protein kinase assays were used to determine whether protein kinases in the elongation zone of the primary root undergo activation or inactivation in response to water deficit. Multiple differences were detected in the phosphoprotein content of root tips of water-stressed compared with wellwatered seedlings. Protein kinase assays identified water-deficitactivated protein kinases, including a 45-kD, Ca2+-independent serine/threonine protein kinase. Water-deficit activation of this kinase occurred within 30 min after transplanting seedlings to conditions of low water potential and was localized to the elongation zone, was independent of ABA accumulation, and was unaffected by cycloheximide-mediated inhibition of protein translation. These results provide evidence that the 45-kD protein kinase acts at an early step in the response of maize primary roots to water deficit and is possibly involved in regulating the adaptation of root growth to low water potential.The accessibility of water is a critica1 environmental determinant of plant growth and distribution. Higher plants possess a variety of mechanisms that may enhance their ability to tolerate water limitation imposed by sporadic rainfall or temperature extremes, and the physiological basis of responses to water deficit has been studied extensively (reviewed by Smith and Griffiths, 1993). However, current understanding of the molecular mechanisms by which plants detect water deficit, transduce that signal to the intracellular machinery, and regulate adaptive responses is limited. Previous work with maize (Zea mays L.) seedlings has shown that shoot and root growth are differentially sensitive to water stress. Whereas shoot elongation decreases to O during moderate water deficit (GW of -0.8 MPa), substantia1 rates of primary root elongation continue under more severe water deficit (GW of -1.6 MPa; Sharp et al., 1988).Furthermore, it has been shown that ABA is involved in the regulation of these growth responses (Saab et al., 1990;Sharp et al., 1994). Because the effects of ABA may be mediated through changes in gene expression (reviewed by Chandler and Robertson, 1994), numerous molecular studies of water stress have focused on identifying gene transcripts that are positively or negatively regulated by ABA and/or water stress. These studies support a model in which multiple mechanisms, including ABA-dependent and -independent pathways, control gene expression in response to water deficit (reviewed by Skriver and Mundy, 1990;Yamaguchi-Shinozaki et al., 1995). However, other experimental approaches may be necessary to identify early-acting signaling molecules, since these are likely to be activated through posttranslational modifications such as reversible phosph...