Molecular characterization and expression pattern of Rubisco activase gene GhRCAß2 in upland cotton (Gossypium hirsutum L.).

ABSTRACT

BACKGROUND: Rubisco 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. OBJECTIVE: To understand the biological function of the GhRCAß2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAß2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern. METHODS: The bioinformatics tools were used to analyze the sequence features of GhRCAß2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAß2 protein. The expression pattern of the GhRCAß2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR). RESULTS: The full-length CDS of GhRCAß2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAß2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAß2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAß2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA ß-isoform. The GhRCAß2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAß2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAß2 in comparison to the wild-type cotton plants. The GhRCAß2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAß2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAß2 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 GhRCAß2 gene. CONCLUSION: Our findings will establish a basis for further understanding the function of the GhRCAß2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.