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

ABSTRACT: A rice cDNA, OsDEP1, encoding a highly cysteine (Cys)-rich G protein ? 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 ? and ? 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.

SUBMITTER: Kunihiro S

PROVIDER: S-EPMC3808331 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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

Kunihiro Shuta S Saito Tatsuhiko T Matsuda Taiki T Inoue Masataka M Kuramata Masato M Taguchi-Shiobara Fumio F Youssefian Shohab S Berberich Thomas T Kusano Tomonobu T

Journal of experimental botany 20131101 14

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 toleran ...[more]

PMID: 24163402

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Project description:BACKGROUND: In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. RESULTS: We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs). In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35?S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. CONCLUSIONS: Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to contribute to gene silencing in leaves because loss of this methylation in synergid cells is associated with CRP gene expression. We discuss this unusual methylation pattern and its alteration in synergid cells as well as the possible retrogene origin and evolutionary significance of CRP genes that are methylated like transposons.

Project description:Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.

Dataset Information

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

Publications

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

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