Molecular characterization and expression pattern of Rubisco activase gene GhRCA2 in upland cotton (Gossypium hirsutum L.)

BackgroundRubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield. ObjectiveTo understand the biological function of the GhRCA beta 2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCA beta 2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern. MethodsThe bioinformatics tools were used to analyze the sequence features of GhRCA beta 2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCA beta 2 protein. The expression pattern of the GhRCA beta 2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR). ResultsThe full-length CDS of GhRCA beta 2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCA beta 2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA alpha-isoform in plants. Evolutionarily, GhRCA beta 2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCA beta 2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA beta-isoform. The GhRCA beta 2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCA beta 2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCA beta 2 in comparison to the wild-type cotton plants. The GhRCA beta 2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCA beta 2 may be regulated by these factors. An additional examination of stress response indicated that GhRCA beta 2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCA beta 2 gene. ConclusionOur findings will establish a basis for further understanding the function of the GhRCA beta 2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.