Molecular Dissection of the SlBAG9 Promoter from Tomato and Its Thermo-Regulatory Activity

Abstract

The Bcl-2-associated athanogene (BAG) gene family plays vital roles in plant growth, development, and biotic and abiotic stress responses. Previous work has demonstrated that tomato SlBAG9, a group II BAG member, negatively regulates plant thermotolerance. However, the regulatory mechanisms governing SlBAG9 expression remain poorly understood. In this study, we isolated and characterized the authentic 1486 bp full-length promoter (P1) of SlBAG9 from the tomato genome. Building upon our previous transcript-level observations, we provide here a detailed functional characterization of this promoter at the cellular and tissue level. In silico analysis identified several key cis-acting regulatory elements, including abscisic acid-responsive elements (ABRE), anaerobic response elements (ARE), and a heat shock element (HSE1). We used stable transgenic tomato plants carrying SlBAG9pro::GUS to verify that the full-length promoter was capable of driving the expression of β-glucuronidase reporter gene (GUS) in transgenic tomato plants, showing GUS staining was detectable in the roots, stems, leaves, flowers, fruits, and seeds, with the highest activity in red-ripe fruits. Notably, GUS activity was significantly upregulated by high temperature (HT) but not by PEG, NaCl, ABA, or cold treatments. To further dissect the HT-responsive regulatory module, we generated three 5′-terminal deletion fragments (−386 bp, P2; −239 bp, P3; and −113 bp, P4) and fused them to GUS. Under HT stress, the smallest deletion P4 showed negligible GUS activity, whereas P1, P2, and P3 retained significant activity. Furthermore, site-directed deletion of the HSE1 element in the full-length context (MU-P1) abolished HT inducibility, confirming that HSE1 serves as a critical positive HT-responsive element. Collectively, these findings confirm and extend our observations that SlBAG9 is a stress-responsive gene, and the characterized HSE1-dependent promoter module represents a promising candidate for genetic engineering aimed at enhancing thermotolerance in crops.