A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants.

By analysing two-dimensional patterns of chloroplastic proteins from Solanum tuberosum, the authors observed the accumulation of a 32-kDa polypeptide in the stroma of plants subjected to water deficit. N-terminus and internal peptides of the protein, named CDSP 32 for chloroplastic drought-induced stress protein, showed no obvious homology with known sequences. Using a serum raised against the protein N-terminus, a cDNA encoding CDSP 32 was cloned by screening an expression library. The deduced mature CDSP 32 protein is 243 amino acids long and displays typical features of thioredoxins in the C-terminal region (122 residues). In particular, CDSP 32 contains a CGPC motif corresponding to a thioredoxin active site and a number of amino acids conferring thioredoxin-type structure. The CDSP 32 C-terminal region was expressed as a fusion protein in Escherichia coli and was shown to possess thioredoxin activity based on reduction assay of insulin disulfide bridges. RNA blot analysis showed that CDSP 32 transcript does not accumulate upon mild water deficit conditions corresponding to leaf relative water contents (RWC) around 85%, but high levels of CDSP 32 transcripts were observed for more severe stress conditions (RWC around 70%). In vivo labelling and immunoprecipitation revealed a substantial increase in CDSP 32 synthesis upon similar stress conditions. Rewatering of wilted plants caused decreases in both transcript and protein abundances. In tomato wild-type plants and ABA-deficient mutants, a similar accumulation of a CDSP 32-related transcript was observed upon water deficit, most likely indicating no requirement for ABA in the regulation of CDSP 32 synthesis. Based on these results, it is proposed that CDSP 32 plays a role in preservation of the thiol: disulfide redox potential of chloroplastic proteins during water deficit.

Characterization of a novel drought-induced 34-kDa protein located in the thylakoids of Solanum tuberosum L. plants.

Using two-dimensional electrophoresis and Coomassie Blue staining, the accumulation of a 34-kDa protein (named cdsp 34 for chloroplastic drought-induced stress protein) is shown in the thylakoids of Solanum tuberosum plants subjected to a progressive and reversible water deficit. In-vivo labeling experiments showed an increased synthesis of cdsp 34 from the early stages of drought stress (leaf relative water content around 85%) and throughout the constraint. Sequences of the N-terminal part and of four tryptic-digest peptides did not reveal significant homology between the cdsp 34 protein and other known proteins. Western blotting analysis, using a serum raised against the N-terminal part of cdsp 34, confirmed the accumulation of cdsp 34 in thylakoids upon drought stress. From immunoblot analysis of different chloroplastic subfractions, the cdsp 34 protein appears to be an extrinsic protein preferentially located in unstacked stroma thylakoids. Immunoprecipitation of in-vitro-translated products, as well as Southern analysis, showed that the cdsp 34 protein is nuclear encoded. After rewatering of water-stressed plants, the level of cdsp 34 synthesis was reduced, but remained substantially higher than in control plants. Western analysis showed the persistence of a high amount of cdsp 34 in rewatered plants for at least two weeks. Based on the abundance and on the location of cdsp 34 within thylakoids, a putative role for this novel chloroplastic protein is discussed in relation to the tolerance of the photosynthetic apparatus of higher plants to dehydration.