Arabidopsis Protein Repair L-Isoaspartyl Methyltransferases: Predominant Activities at Lethal Temperatures

Protein l -isoaspartyl ( d -aspartyl) O -methyltransferases (Enzyme Commission (EC) 2.1.1.77; PIMT or PCMT) are enzymes that initiate the full or partial repair of damaged l -aspartyl and l -asparaginyl residues, respectively. These enzymes are found in most organisms and maintain a high degree of sequence conservation. Arabidopsis thaliana ( Arabidopsis L. Heynh.) is unique among eukaryotes in that it contains two genes, rather than one, that encode PIMT isozymes. We describe a novel A. thaliana PIMT isozyme, designated At PIMT2αω, encoded by the PIMT2 gene ( At5g50240 ). We characterized the enzymatic activity of the recombinant At PIMT2αω in comparison to the other At PIMT2 isozymes, At PIMT1, and to the human PCMT1 ortholog, to better understand its role in Arabidopsis . All Arabidopsis PIMT isozymes are active over a relatively wide pH range. For At PIMT2αω maximal activity is observed at 50°C (a lethal temperature for Arabidopsis ); this activity is almost 10 times greater than the activity at the growth temperature of 25°C. Interestingly, enzyme activity decreases after pre-incubation at temperatures above 30°C. A similar situation is found for the recombinant At PIMT2ψ and the At PIMT2ω isozymes, as well as for the At PIMT1 and human PCMT1 enzymes. These results suggest that the short-term ability of these methyltransferases to initiate repair under extreme temperature conditions may be a common feature of both the plant and animal species.     

Plastids contain a second sec translocase system with essential functions

Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis

As in mammalian systems, heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are present in plants and are involved in the regulation of development and cell signaling. Besides the sole prototypical G protein alpha subunit gene, GPA1, the Arabidopsis thaliana genome has three extra-large GTP-binding protein (XLG)-encoding genes: XLG1 (At2g23460), XLG2 (At4g34390) and XLG3 (At1g31930). The C-termini of the XLGs are Galpha domains that are homologous to GPA1, whereas their N-termini each contain a cysteine-rich region and a putative nuclear localization signal (NLS). GFP fusions with each XLG confirmed nuclear localization. All three XLG genes are expressed in essentially all plant organs, with strong expression in vascular tissues, primary root meristems and lateral root primordia. Analysis of single, double and triple T-DNA insertional mutants of the XLG genes revealed redundancy in XLG function. Dark-grown xlg1-1 xlg2-1 xlg3-1 triple mutant plants showed markedly increased primary root length compared with wild-type plants. This phenotype was not observed in dark-grown xlg single mutants, and was suppressed upon complementation of the xlg triple mutant with each XLG. Root cell sizes of the xlg triple mutant and root morphology were highly similar to those of wild-type roots, suggesting that XLGs may regulate cell proliferation. Dark-grown roots of the xlg triple mutants also showed altered sensitivity to sugars, ABA hyposensitivity and ethylene hypersensitivity, whereas seed germination in xlg triple mutants was hypersensitive to osmotic stress and ABA. As plant-specific proteins, regulatory mechanisms of XLGs may differ from those of conventional Galphas.     

Selective inhibition of clade A phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors

Clade A protein phosphatases type 2C (PP2Cs) are negative regulators of abscisic acid (ABA) signaling that are inhibited in an ABA-dependent manner by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) intracellular receptors. We provide genetic evidence that a previously uncharacterized member of this PP2C family in Arabidopsis (Arabidopsis thaliana), At5g59220, is a negative regulator of osmotic stress and ABA signaling and that this function was only apparent when double loss-of-function mutants with pp2ca-1/ahg3 were generated. At5g59220-green fluorescent protein and its close relative PP2CA-green fluorescent protein showed a predominant nuclear localization; however, hemagglutinin-tagged versions were also localized to cytosol and microsomal pellets. At5g59220 was selectively inhibited by some PYR/PYL ABA receptors, and close relatives of this PP2C, such as PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (AHG3) and AHG1, showed a contrasting sensitivity to PYR/PYL inhibition. Interestingly, AHG1 was resistant to inhibition by the PYR/PYL receptors tested, which suggests that this seed-specific phosphatase is still able to regulate ABA signaling in the presence of ABA and PYR/PYL receptors and therefore to control the highly active ABA signaling pathway that operates during seed development. Moreover, the differential sensitivity of the phosphatases At5g59220 and PP2CA to inhibition by ABA receptors reveals a functional specialization of PYR/PYL ABA receptors to preferentially inhibit certain PP2Cs.     

The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.