Comparing four heat-inducible promoters in stably transformed sugarcane regarding spatial and temporal control of transgene expression reveals candidates to drive stem-preferred transgene expression

Small heat shock protein (sHSP) promoters contain <i>cis</i>-regulatory elements that facilitate transcription in response to heat stress, making them valuable tools for functional studies through controlled gene expression and the precise regulation of gene-editing tools or morphogenic regulators. To evaluate their utility, GUS reporter gene expression driven by four plant-sourced <i>HSP</i> promoters (p<i>GmHSP17.5</i>, p<i>HvHSP17</i>, p<i>ZmHSP17.7</i>, and p<i>ZmHSP26</i>) was compared across various tissues of stably transformed sugarcane before and after heat treatment. At 22°C, all promoters showed minimal activity in leaves and roots, although p<i>ZmHSP17.7</i> and p<i>HvHSP17</i> displayed moderate expression in stems. Following heat treatment, all promoters exhibited their highest activity in stems, followed by leaves and roots. In stem tissues, p<i>GmHSP17.5</i> displayed heat-induced <i>uidA</i> expression comparable to the constitutive p<i>ZmUbi</i> promoter. Notably, heat-induced reporter gene activity in stem middle sections of single-copy transgenic lines containing p<i>ZmHSP17.7</i>, p<i>HvHSP17</i>, or p<i>ZmHSP26</i> exceeded p<i>ZmUbi</i>-derived <i>uidA</i> activity by 9.7-fold, 3.8-fold, and 3.0-fold, respectively, with 346- to 3,672-fold induction compared to control conditions. Most promoters showed peak expression in the middle sections of the stem, while p<i>HvHSP17</i> was the most active in the stem apices. Histochemical analysis revealed that p<i>ZmHSP17.7</i> and p<i>HvHSP17</i> were active in both parenchyma cells and vascular bundles within sugarcane stems. Among leaf tissues, mature leaves exhibited greater expression than senescing or immature leaves, while root activity remained consistently minimal across all promoters. Temperature-course experiments identified distinct activation thresholds: 34°C-36°C for p<i>ZmHSP17.7</i>, 36°C for p<i>ZmHSP26</i>, 36°C-38°C for p<i>HvHSP17</i>, and 40°C-42°C for p<i>GmHSP17.5</i>. Drought stress also induced reporter gene transcription in stems under <i>HSP</i> promoters, although with lower fold induction than heat treatment. These findings provide valuable tools for gene function studies and biotechnology applications, including heat stress tolerance research, controlled transgene expression in metabolic engineering, precision gene editing, and developmental biology studies.