Overexpression of a new proline-rich protein encoding Gene CsPRP4 increases starch accumulation in Citrus

Proline-rich proteins (PRPs) are a group of proteins with repeated proline/hydroproline residues that synthesize cell walls. Here, we identified CsPRP4, a new gene encoding a hybrid PRP, from Citrus sinensis. CsPRP4 showed preferential expression in leaves and significant upregulation when treated with exogenous ethylene. We used the Citrus sinensis (L.) Osbeck genome to clone the 1.5 kb CsPRP4 promoter and performed a GUS activity assay. The CsPRP4 promoter drove GUS expression in the leaf tissue of Arabidopsis thaliana transgenic plants in response to external stimuli. To functionally characterize CsPRP4, Citrus transformation was conducted using the full-length CsPRP4 cDNA sense sequence with a binary vector. Electron microscopy and physiological assays revealed that overexpression of CsPRP4 increased the number and size of starch granules in the chloroplast. RNA-Seq analysis identified 467 genes in transgenic plants that were significantly differentially expressed compared with the WT. Among all differentially expressed genes (DEGs), key genes encoding starch biosynthetic enzymes were upregulated in transgenic plants, while starch degradation-related genes were downregulated. Taken together, these results demonstrate a potential role of CsPRP4 in leaf development and sugar metabolism.