Functional Characterization of ClHSP22.9 Promoter from Wild Watermelon (Citrullus lanatus) Reveals Heat- and Drought-Inducible Transcriptional Activity
- 1Department of Horticulture, Zhejiang University, Hangzhou 310058, China
- 2Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar 25130, Pakistan
- 3Department of Biotechnology, Abdul Wali Khan University, Mardan 23200, Pakistan
Abstract
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.
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BibTeX
@article{munir2026functional,
title = {{Functional Characterization of ClHSP22.9 Promoter from Wild Watermelon (Citrullus lanatus) Reveals Heat- and Drought-Inducible Transcriptional Activity}},
author = {Raheel Munir and Iqbal Hussain and Nazia Jan and Bahar Ali and Iqbal Munir and Waqar Ali and Raham Sher Khan and Aqib Iqbal},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27219648},
url = {https://www.mdpi.com/1422-0067/27/21/9648},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Functional Characterization of ClHSP22.9 Promoter from Wild Watermelon (Citrullus lanatus) Reveals Heat- and Drought-Inducible Transcriptional Activity
AU - Raheel Munir
AU - Iqbal Hussain
AU - Nazia Jan
AU - Bahar Ali
AU - Iqbal Munir
AU - Waqar Ali
AU - Raham Sher Khan
AU - Aqib Iqbal
PY - 2026
DA - 2026/10/28
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219648
UR - https://www.mdpi.com/1422-0067/27/21/9648
AB - 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.
SN - 1422-0067
ER -