Dataset Information

Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.

ABSTRACT:

Background

The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl^-). Thus Cl^- influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na⁺) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl^- and nitrate (NO₃^-), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl^- and NO₃^- homeostasis under salt stress.

Results

Among the eight soybean CLC genes, the tonoplast-localized c2 has uniquely different transcriptional patterns between cultivated soybean N23674 and wild soybean BB52. Using soybean hairy root transformation, we found that GsCLC-c2 over-expression contributed to Cl^- and NO₃^- homeostasis, and therefore conferred salt tolerance, through increasing the accumulation of Cl^- in the roots, thereby reducing their transportation to the shoots where most of the cellular damages occur. Also, by keeping relatively high levels of NO₃^- in the aerial part of the plant, GsCLC-c2 could reduce the Cl^-/NO₃^- ratio. Wild type GsCLC-c2, but not its mutants (S184P, E227V and E294G) with mutations in the conserved domains, is able to complement Saccharomyces cerevisiae △gef1 Cl^- sensitive phenotype. Using two-electrode voltage clamp on Xenopus laevis oocytes injected with GsCLC-c2 cRNA, we found that GsCLC-c2 transports both Cl^- and NO₃^- with slightly different affinity, and the affinity toward Cl^- was pH-independent.

Conclusion

This study revealed that the expression of GsCLC-c2 is induced by NaCl-stress in the root of wild soybean. The tonoplast localized GsCLC-c2 transports Cl^- with a higher affinity than NO₃^- in a pH-independent fashion. GsCLC-c2 probably alleviates salt stress in planta through the sequestration of excess Cl^- into the vacuoles of root cells and thus preventing Cl^- from entering the shoots where it could result in cellular damages.

SUBMITTER: Wei P

PROVIDER: S-EPMC6444504 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Publications

Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.

Wei Peipei P Che Benning B Shen Like L Cui Yiqing Y Wu Shengyan S Cheng Cong C Liu Feng F Li Man-Wah MW Yu Bingjun B Lam Hon-Ming HM

BMC plant biology 20190401 1

<h4>Background</h4>The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl-). Thus Cl- influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na+) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl- and nitrate (NO3-), a ...[more]

PMID: 30935372

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Project description:Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. The bone building cells, osteoblasts, are derived from mesenchymal stromal cells (MSCs); however, with increasing age osteogenic differentiation is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes involved in osteoblast differentiation of human bone marrow-derived MSCs (hMSCs). We identified the gene chloride intracellular channel protein 3 (CLIC3) to be strongly upregulated during MSC-derived osteoblast differentiation. Lentiviral overexpression of CLIC3 in hMSCs caused a 60% increase of matrix mineralization. Conversely, knockdown of CLIC3 in hMSCs using two short-hairpin RNAs (shRNAs) against CLIC3 resulted in a 69% to 76% reduction in CLIC3 mRNA expression, 53% to 37% less alkaline phosphatase (ALP) activity, and 78% to 88% less matrix mineralization compared to scrambled control. Next, we used an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15-fold increase in bone formation (0.33% versus 5.05% bone area relative to scaffold). Using a Clic3-His-tagged pull-down assay and liquid chromatography-mass spectrometry (LS/MS)-based proteomics analysis in lysates of osteogenically differentiated hMSCs, we showed that CLIC3 interacts with NIMA-related kinase 9 (NEK9) and phosphatidylserine synthase 1 (PTDSS1) in vitro, and this finding was supported by immunofluorescent analysis. In addition, inhibition of NEK9 or PTDSS1 gene expression by shRNAs inhibited osteoblast differentiation and mineralization. In conclusion, we successfully identified CLIC3 to be a lineage-specific gene regulating osteoblast differentiation and bone formation through its interaction with NEK9 and PTDSS1. © The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Dataset Information

Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.

Background

Results

Conclusion

Publications

Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.

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