GhDLDH1 up-regulates synthesis of chloroplast responding to salinity stress in Gossypium hirsutum L.

Abstract

D-lactate dehydrogenase (D-LDH) is a key enzyme in the pyruvate metabolic pathway and is crucial for plant growth, development, and stress tolerance. This study provides a systematic analysis of the genetic characteristics, evolutionary history, cis-acting elements, and expression patterns of the D-LDH gene family in cotton with 14 D-LDH genes were discovered. Phylogenetic analysis showed that D-LDH family genes could be clustered into 2 clades. The D-LDH genes of the four cotton species show a closer relationship to those of T. cacao compared to other plant species. Structure analysis showed that GhDLDH genes from the same subgroup exhibited similar genetic structure. The D-LDH family members predominantly include elements associated with hormones, the light response, the drought response, stress and the temperature response. GhDLDH1 was significantly upregulated following exposure to various stress treatments. The quantitative real-time PCR (qRT-PCR) results demonstrated that most GhDLDH genes responded positively to salt stress, with GhDLDH1 exhibited the peak transcriptional activity. Subcellular localization analysis revealed that GhDLDH1 is specifically localized within the chloroplast. Compared with those of pYL156 plants, pYL156: GhDLDH1 plants presented significantly reductions in pyruvate (PA), lactic acid (LA), glutathione (GSH), lactic dehydrogenase (LDH), and chlorophyll levels, with a significant increase in the methylglyoxal (MG) content under salt stress. GhDLDH1 gene silencing in cotton seedlings resulted in heightened sensitivity to NaCl stress, as evidenced by trypan blue staining. Plants in which the GhDLDH1 gene was silenced exhibited lower chloroplast levels compared with pYL156 plants, and the structure was less coherent. Potential interactions were revealed between the D-LDH protein and the chloroplast-associated proteins PKP1 and PKP3. And the qRTPCR data revealed altered expression levels of GhPKP1 and GhPKP3 following GhDLDH1 gene silencing. In conjunction with the observed changes in chloroplast number and morphology, these findings suggest that GhDLDH1 may be a key regulator of chloroplast function. This study serves as a valuable reference for investigating the interaction between the D-LDH gene and chloroplast-associated genes, as well as their regulatory effects on cotton salt tolerance.