Identification of transcriptional regulators interacting with PEPC genes and characterization of bHLH49 in Suaeda aralocaspica.

Abstract

The unique single-cell C₄ photosynthetic pathway of Suaeda aralocaspica has garnered significant attention owing to its specialized photosynthetic structure and the relevant enzymes, e.g., the phosphoenolpyruvate carboxylase (PEPC). However, the regulatory mechanisms controlling the transcriptional activity of SaPEPC1 and SaPEPC2 and their promoters under environmental stress conditions remain poorly understood. Here, we aimed to identify and characterize factors involved in the transcriptional regulation of these SaPEPC gene promoters using a yeast-one-hybrid system. We constructed a cDNA library of S. aralocaspica under salt stress, achieving a recombination efficiency of 100 % with insert lengths of 200-2000 bp and a library capacity of 1.2 × 10⁷ CFU·mL^-1. Screening results revealed 75 and 74 candidate regulatory factors interacting with the SaPEPC1 and SaPEPC2 promoters (groups 1 and 2), respectively. Group 1 factors were enriched in photosynthesis-related events, whereas group 2 predominantly included stress-responsive factors. Further analyses indicated significantly higher transcriptional responsiveness of factors in group 1 factors (especially the SaPEPC1 gene itself) than group 2 factors under salt stress, osmotic stress (PEG), and light/dark conditions. Notably, SabHLH49 from group 1 distinctly enhanced GUS activity driven by the SaPEPC1 promoter; three-dimensional structure prediction and dual-luciferase assay confirmed the potential interaction between SabHLH49 and the SaPEPC1 promoter. The specific interaction region potentially involved three predicted motifs: "GACACATGTA," "GTCACGAACA," and "ATCTCATGCG." Transgenic Arabidopsis overexpressing SabHLH49 showed enhanced salt tolerance, likely by alleviating cell membrane damage and enhancing photosynthetic efficiency under salt stress. Our findings suggest that SabHLH49-PEPC1 interaction may impact PEPC regulation, which improves our understanding of the mechanisms regulating photosynthesis-associated events.