Functional Characterization of ClHSP22.9 Promoter from Wild Watermelon (Citrullus lanatus) Reveals Heat- and Drought-Inducible Transcriptional Activity

Small heat shock proteins (sHSPs) are essential components of the plant proteostasis network under environmental stress; however, the cis-regulatory mechanisms underlying stress-responsive expression of individual sHSP genes remains insufficiently depicted. Here, we characterized the promoter of ClHSP22.9 from wild watermelon (Citrullus lanatus subsp. cordophanus) and evaluated its transcriptional responsiveness under different abiotic stresses. A semi-quantitative RT-PCR analysis revealed that ClHSP22.9 transcripts were strongly induced by heat, dehydration, and salinity treatments, whereas cold treatment caused comparatively limited transcriptional alterations. A 1482 bp promoter region upstream of the transcription start site was isolated using thermal asymmetric interlaced PCR (TAIL-PCR) and partially overlapping primer-based PCR (POP-PCR). An in silico promoter analysis identified multiple predicted stress and hormonal-associated cis-regulatory elements, including two heat shock elements (HSEs). Three serial 5′ promoter deletion fragments (−1482, −1012, and −522 bp) were fused to the GUS reporter gene and functionally analyzed in transgenic Brassica napus. Under heat stress, the −1482 and −1012 bp promoter fragments exhibited strong reporter activation, whereas deletion to −522 bp significantly reduced promoter activity, indicating that the −1012 to −522 bp region contributes substantially to maximal heat-responsive transcription. Furthermore, the −1012 bp proHSPII fragment retained an inducible GUS expression under heat, drought, and combined heat and drought stress conditions. These findings identify an important region within the ClHSP22.9 promoter and establish the −1012 bp promoter fragment as a promising candidate stress-responsive regulatory sequence for further mechanistic characterization and potential application in plant biotechnology.