Characterization of the MeWRKY12–MeSSIV Regulatory Module and MeWRKY12 Promoter Activity in Cassava

Background: Soluble starch synthases are essential for starch formation, but their transcriptional regulation remains poorly understood in cassava. Starch synthase IV (SSIV) is an important enzyme in starch biosynthesis, and this study aimed to investigate the transcriptional regulation of cassava MeSSIV. Methods: Yeast one-hybrid, transcriptional activation, subcellular localization, and dual luciferase assays were performed to evaluate MeWRKY12 binding to and activation of the MeSSIV promoter. Quantitative real-time PCR was used to analyze MeWRKY12 expression in different tissues. Histochemical staining and quantitative GUS assays were conducted using proMeWRKY12 reporter plants grown under field conditions for six months. Promoter responses to abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA3) were examined in storage root slices. Results: MeWRKY12 bound to the tested MeSSIV promoter region in yeast, exhibited transcriptional activation activity, localized to the nucleus, and enhanced MeSSIV promoter activity in plant cells. MeWRKY12 transcript abundance was highest in storage roots. GUS activity was also highest in storage roots, followed by fibrous roots. Histochemical staining revealed broad promoter activity in vascular, cambial, and parenchymatous tissues, including the xylem parenchyma of storage roots. Promoter activity increased following ABA, MeJA, and GA3 treatments, with distinct temporal response patterns. Conclusions: These findings provide evidence at the promoter level supporting MeWRKY12 as a candidate positive regulator of MeSSIV transcription and reveal variation in proMeWRKY12 activity across tissues and hormone treatments. This study provides a basis for investigating WRKY transcription factors in the regulation of starch biosynthesis in cassava.