Chloroplast translation factor EF-Tu of Arabidopsis thaliana can be inactivated via oxidation of a specific cysteine residue.

Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.

Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum Can Be Estimated by Non-Photochemical Quenching upon Actinic Illumination During the Dark Period.

Mesembryanthemum crystallinum, which switches the mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) upon high salt stress, was shown here to exhibit diurnal changes in not only the CO2 fixation pathway but also Chl fluorescence parameters under CAM-induced conditions. We conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. We found that the diurnal change in the status of electron transfer could be ascribed to the formation of a proton gradient across thylakoid membranes presumably resulting from diurnal changes in the ATP/ADP ratio reported earlier. The electron transport by actinic illumination thus became limited at the step of plastoquinol oxidation by the Cyt b6/f complex in the 'night' period upon CAM induction, resulting in high levels of non-photochemical quenching. The actinically induced non-photochemical quenching in the 'night' period correlated well with the degree of CAM induction. Chl fluorescence parameters, such as NPQ or qN, could be used as a simple indexing system for the CAM induction.

Characterization of the plastidic phosphate translocators in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum.

In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.

Isolation and characterization of a polyubiquitin gene and its promoter region from Mesembryanthemum crystallinum.

Transcript levels of the polyubiquitin gene McUBI1 had been reported to be constant during Crassulacean acid metabolism (CAM) induction in the facultative CAM plant, Mesembryanthemum crystallinum. Here, we report the sequences of the full-length cDNA of McUBI1 and its promoter, and validation of the McUBI1 promoter as an internal control driving constitutive expression in transient assays using the dual-luciferase system to investigate the regulation of CAM-related gene expression. The McUBI1 promoter drove strong, constitutive expression during CAM induction. We compared the activities of this promoter with those of the cauliflower mosaic virus (CaMV) 35S promoter in detached C3- and CAM-performing M. crystallinum and tobacco leaves. We confirmed stable expression of the genes controlled by the McUBI1 promoter with far less variability than under the CaMV 35S promoter in M. crystallinum, whereas both promoters worked well in tobacco. We found the McUBI1 promoter more suitable than the CaMV 35S promoter as an internal control for transient expression assays in M. crystallinum.

Transcriptional profiles of organellar metabolite transporters during induction of crassulacean acid metabolism in Mesembryanthemum crystallinum.

Metabolite transport across multiple organellar compartments is essential for the operation of crassulacean acid metabolism (CAM). To investigate potential circadian regulation of inter-organellar metabolite transport processes, we have identified eight full-length cDNAs encoding an organellar triose phosphate / Pi translocator (McTPT1), a phosphoenolpyruvate / Pi translocator (McPPT1), two glucose-6-phosphate / Pi translocators (McGPT1, 2), two plastidic Pi translocator-like proteins (McPTL1, 2), two adenylate transporters (McANT1, 2), a dicarboxylate transporter (McDCT2), and a partial cDNA encoding a second dicarboxylate transporter (McDCT1) in the model CAM plant, Mesembryanthemum crystallinum L. We next investigated day / night changes in steady-state transcript abundance of each of these transporters in plants performing either C3 photosynthesis or CAM induced by salinity or water-deficit stress. We observed that the expression of both isogenes of the glucose-6-phosphate / Pi translocator (McGPT1, 2) was enhanced by CAM induction, with McGPT2 transcripts exhibiting much more pronounced diurnal changes in transcript abundance than McGPT1. Transcripts for McTPT1, McPPT1, and McDCT1 also exhibited more pronounced diurnal changes in abundance in the CAM mode relative to the C3 mode. McGPT2 and McDCT1 transcripts exhibited sustained oscillations for at least 3 d under constant light and temperature conditions suggesting their expression is under circadian clock control. McTPT1 and McGPT2 transcripts were preferentially expressed in leaf tissues in either C3 or CAM modes. The leaf-specific and / or circadian controlled gene expression patterns are consistent with McTPT1, McGPT2 and McDCT1 playing CAM-specific metabolite transport roles.

Transcript profiling of salinity stress responses by large-scale expressed sequence tag analysis in Mesembryanthemum crystallinum.

The common ice plant, Mesembryanthemum crystallinum, is a halophytic (salt-loving) member of the Aizoaceae, which switches from C3 photosynthesis to Crassulacean acid metabolism (CAM) when exposed to salinity or water-deficit stress. CAM is a metabolic adaptation of photosynthetic carbon fixation that improves water use efficiency by shifting net CO2 uptake to the night, thereby reducing transpirational water loss. To improve our understanding of the molecular genetic underpinnings and control mechanisms for Crassulacean acid metabolism (CAM) and other salinity stress response adaptations, a total of 9733 expressed sequence tags (ESTs) from cDNAs derived from leaf tissues of well-watered and salinity-stressed (0.5 M NaCl for 30 and 48 h) were characterized. Clustering and assembly of these ESTs resulted in the identification of a total of 3676 tentative unique gene sequences (1249 tentative consensus sequences and 2427 singleton ESTs) expressed in leaves of ice plant under unstressed and salinity stressed conditions. The same number (2782) of ESTs from each library (total=8346 ESTs) were randomly selected and analyzed to compare expression profiles among the control and salt stressed leaf tissues. EST frequencies for transcripts encoding CAM-related enzymes, pathogenesis-related, senescence-associated, cell death-related, and stress-related proteins such as heat shock proteins (HSPs), chaperones, early light-inducible proteins, ion homeostasis, antioxidative stress, detoxification, and biosynthetic enzymes for osmoprotectants increased 2-12-fold in cDNA libraries constructed from salt stressed plants. In contrast, the frequency of ESTs encoding light-harvesting and photosystem complexes and C3 photosynthetic enzymes decreased 4-fold overall following salinity stress with transcripts for ribulose bisphosphate carboxylase/oxygenase (RuBisCO) subunits decreasing 7-fold. Moreover, stressed plants contained a higher percentage of ESTs encoding novel and/or functionally unknown proteins. The rapid discovery of both known and unknown genes related to stress responses in M. crystallinum demonstrates the great utility of EST analysis in unraveling the complex set of adaptive mechanisms contributing to water use efficiency (CAM) and salinity tolerance.