Redox Regulation of Monodehydroascorbate Reductase by Thioredoxin y in Plastids Revealed in the Context of Water Stress.

Thioredoxins (TRXs) are key players within the complex response network of plants to environmental constraints. Here, the physiological implication of the plastidial y-type TRXs in Arabidopsis drought tolerance was examined. We previously showed that TRXs y1 and y2 have antioxidant functions, and here, the corresponding single and double mutant plants were studied in the context of water deprivation. TRX y mutant plants showed reduced stress tolerance in comparison with wild-type (WT) plants that correlated with an increase in their global protein oxidation levels. Furthermore, at the level of the main antioxidant metabolites, while glutathione pool size and redox state were similarly affected by drought stress in WT and trxy1y2 plants, ascorbate (AsA) became more quickly and strongly oxidized in mutant leaves. Monodehydroascorbate (MDA) is the primary product of AsA oxidation and NAD(P)H-MDA reductase (MDHAR) ensures its reduction. We found that the extractable leaf NADPH-dependent MDHAR activity was strongly activated by TRX y2. Moreover, activity of recombinant plastid Arabidopsis MDHAR isoform (MDHAR6) was specifically increased by reduced TRX y, and not by other plastidial TRXs. Overall, these results reveal a new function for y-type TRXs and highlight their role as major antioxidants in plastids and their importance in plant stress tolerance.

Plastid-cytosol partitioning and integration of metabolic pathways for APS/PAPS biosynthesis in Arabidopsis thaliana.

Plants assimilate sulfate from the environment to synthesize biologically active sulfur-containing compounds required for growth and cellular development. The primary steps of sulfur metabolism involve sequential enzymatic reactions synthesizing adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recent finding suggests that an adenosine nucleotide transport system facilitating the exchange of PAPS and 3'-phosphoadenosine 5'-phosphate across the plastid envelope is essential for establishing an intimate connection between the plastidic and cytosolic sulfate assimilation pathways in plants. Subcellular partitioning and integration of metabolic pathways provide focal points for investigating metabolic flux regulations. This perspective article presents an integrative view of sulfur metabolic flux control mechanisms with an emphasis on subcellular partitioning of APS/PAPS biosynthetic pathways in Arabidopsis thaliana.

Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana.

Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS) is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3, and -4) encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUG(Met1) to produce plastid-targeted ATPS2 pre-proteins or at AUG(Met52) or AUG(Met58) within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUG(Met52) or AUG(Met58) was verified by expressing a tandem-fused synthetic gene, ATPS2 (5'UTR-His12) :Renilla luciferase:ATPS2 (Ile13-Val77) :firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUG(Met52) and AUG(Met58) significantly reduced the firefly luciferase activity, while AUG(Met52) was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUG(Met52) or AUG(Met58) was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.

New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

Intracellular Ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana.

In Arabidopsis thaliana cell suspension, abscisic acid (ABA) induces changes in cytosolic calcium concentration ([Ca(2+)](cyt)) which are the trigger for ABA-induced plasma membrane anion current activation, H(+)-ATPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca(2+) stores in ABA signal transduction through electrophysiological current measurements, cytosolic Ca(2+) activity measurements with the apoaequorin Ca(2+) reporter protein and external pH measurement. Intracellular Ca(2+) stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor (U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (1) Ca(2+) release in the cytosol, (2) anion channel activation and H(+)-ATPase inhibition leading to plasma membrane depolarization and (3) stomatal closure. Intracellular Ca(2+) release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.