Shohab Youssefian

Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation

By means of functional screening using the cadmium (Cd)-sensitive ycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1, from Digitaria ciliaris growing in a former mining area in northern Japan, and have shown that it confers Cd tolerance to the yeast cells, which accumulated almost 2-fold lower Cd levels than control cells. The 521 bp DcCDT1 cDNA contains an open reading frame of 168 bp and encodes a deduced peptide, DcCDT1, that is 55 amino acid residues in length, of which 15 (27.3%) are cysteine residues. Five DcCDT1 homologs (here termed OsCDT1-OsCDT5) have been identified in rice, and all of them were up-regulated to varying degrees in the above-ground tissues by CdCl(2) treatment. Localization of green fluorescent protein fusions suggests that DcCDT1 and OsCDT1 are targeted to both cytoplasmic membranes and cell walls of plant cells. Transgenic Arabidopsis thaliana plants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerant phenotype and, consistent with our yeast data, accumulated lower amounts of Cd when grown on CdCl(2). Collectively, our data suggest that DcCDT1 and OsCDT1 function to prevent entry of Cd into yeast and plant cells and thereby enhance their Cd tolerance.

Rice DEP1, encoding a highly cysteine-rich G protein γ subunit, confers cadmium tolerance on yeast cells and plants

A rice cDNA, OsDEP1, encoding a highly cysteine (Cys)-rich G protein γ subunit, was initially identified as it conferred cadmium (Cd) tolerance on yeast cells. Of the 426 aa constituting OsDEP1, 120 are Cys residues (28.2%), of which 88 are clustered in the C-terminal half region (aa 170–426). To evaluate the independent effects of these two regions, two truncated versions of the OsDEP1-expressing plasmids pOsDEP1(1–169) and pOsDEP1(170–426) were used to examine their effects on yeast Cd tolerance. Although OsDEP1(170–426) conferred a similar level of Cd tolerance as the intact OsDEP1, OsDEP1(1–169) provided no such tolerance, indicating that the tolerance effect is localized to the aa 170–426 C-terminal peptide region. The Cd responses of transgenic Arabidopsis plants constitutively expressing OsDEP1, OsDEP1(1–169) or OsDEP1(170–426), were similar to the observations in yeast cells, with OsDEP1 and OsDEP1(170–426) transgenic plants displaying Cd tolerance but OsDEP1(1–169) plants showing no such tolerance. In addition, a positive correlation between the transcript levels of OsDEP1 or OsDEP1(170–426) in the transgenics and the Cd content of these plants upon Cd application was observed. As several Arabidopsis loss-of-function heterotrimeric G protein β and γ subunit gene mutants did not show differences in their Cd sensitivity compared with wild-type plants, we propose that the Cys-rich region of OsDEP1 may function directly as a trap for Cd ions.

A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants

We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3 %) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1~5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus tremula x Populus alba) and Picea sitchensis, appear to possess only a single homologue. GFP fusion experiments demonstrate that DcCDT1 and OsCDT1 are targeted to both the plant cytoplasmic membranes and cell walls. Constitutive expression of DcCDT1 or OsCDT1 confers Cd-tolerance to transgenic A. thaliana plants by lowering the accumulation of Cd in the cells. The functions of the DcCDT1 family members are discussed in the light of these findings.

Overexpression of rice OsREX1 - S , encoding a putative component of the core general transcription and DNA repair factor IIH, renders plant cells tolerant to cadmium- and UV-induced damage by enhancing DNA excision repair

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.

Manipulation of Genes Involved in Sulfur and Glutathione Metabolism

Sulfur is an essential nutrient for plant growth and development and, under conditions of sufficient sulfur availability, is taken up from the soil by roots in the form of sulfate and transferred to the shoots. The sulfate is subsequently activated and then reduced to sulfite and then to sulfide, in what is generally referred to as the sulfur reduction assimilatory pathway; the main function and ultimate product of which is the biosynthesis of cysteine (Cys). The Cys thus formed serves as a precursor for several other reduced sulfur-containing metabolites, notably methionine and glutathione (GSH), the latter constituting the principal storage and transport form of reduced sulfur in plants, as well as a key factor controlling plant responses to a variety of biotic and abiotic stresses (Noctor et al. 1998a; May et al. 1998a).