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

Qiande, Moni; Altpeter, Fredy

doi:10.3389/fpls.2025.1709171

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

Journal Article · Tue Dec 02 23:00:00 EST 2025 · Frontiers in Plant Science

DOI:https://doi.org/10.3389/fpls.2025.1709171· OSTI ID:3006325

Qiande, Moni ^[1]; Altpeter, Fredy ^[1]

Univ. of Florida, Gainesville, FL (United States); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Small heat shock protein (sHSP) promoters contain cis-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 HSP promoters (pGmHSP17.5, pHvHSP17, pZmHSP17.7, and pZmHSP26) 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 pZmHSP17.7 and pHvHSP17 displayed moderate expression in stems. Following heat treatment, all promoters exhibited their highest activity in stems, followed by leaves and roots. In stem tissues, pGmHSP17.5 displayed heat-induced uidA expression comparable to the constitutive pZmUbi promoter. Notably, heat-induced reporter gene activity in stem middle sections of single-copy transgenic lines containing pZmHSP17.7, pHvHSP17, or pZmHSP26 exceeded pZmUbi-derived uidA 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 pHvHSP17 was the most active in the stem apices. Histochemical analysis revealed that pZmHSP17.7 and pHvHSP17 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 pZmHSP17.7, 36°C for pZmHSP26, 36°C–38°C for pHvHSP17, and 40°C–42°C for pGmHSP17.5. Drought stress also induced reporter gene transcription in stems under HSP 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Illinois at Urbana-Champaign, IL (United States); Univ. of Florida, Gainesville, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDA National Institute of Food and Agriculture

Grant/Contract Number:: SC0018420

OSTI ID:: 3006325

Journal Information:: Frontiers in Plant Science, Journal Name: Frontiers in Plant Science Vol. 16; ISSN 1664-462X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (67)

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. Jefferson, R. A.; Kavanagh, T. A.; Bevan, M. W. The EMBO Journal, Vol. 6, Issue 13 https://doi.org/10.1002/j.1460-2075.1987.tb02730.x	journal	December 1987
Heat‐shock‐inducible CRISPR/Cas9 system generates heritable mutations in rice Nandy, Soumen; Pathak, Bhuvan; Zhao, Shan Plant Direct, Vol. 3, Issue 5 https://doi.org/10.1002/pld3.145	journal	May 2019
Alfalfa heat shock genes are differentially expressed during somatic embryogenesis Gy�rgyey, J�nos; Gartner, Anton; N�meth, Kinga Plant Molecular Biology, Vol. 16, Issue 6 https://doi.org/10.1007/BF00016072	journal	June 1991
Development of a heat shock inducible expression cassette for plants: Characterization of parameters for its use in transient expression assays Ainley, W. Michael; Key, Joe L. Plant Molecular Biology, Vol. 14, Issue 6 https://doi.org/10.1007/BF00019392	journal	June 1990
Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter Coca, Maria A.; Almoguera, Concepci�n; Thomas, Terry L. Plant Molecular Biology, Vol. 31, Issue 4 https://doi.org/10.1007/BF00019473	journal	July 1996
Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants Pr�ndl, Ralf; Kloske, Eva; Sch�ffl, Fritz Plant Molecular Biology, Vol. 28, Issue 1 https://doi.org/10.1007/BF00042039	journal	April 1995
Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes Matzke, A. J. M.; Neuhuber, F.; Park, Y. D. Molecular and General Genetics MGG, Vol. 244, Issue 3 https://doi.org/10.1007/BF00285449	journal	May 1994
TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants Xue, Hua; Yang, Yu-Tao; Wu, Chang-Ai Theoretical and Applied Genetics, Vol. 110, Issue 4 https://doi.org/10.1007/s00122-004-1880-9	journal	January 2005
Specimen block counter-staining for localization of GUS expression in transgenic arabidopsis and tobacco M., Kim; J. -W., Choi; J. -H., Jeon Plant Cell Reports, Vol. 21, Issue 1 https://doi.org/10.1007/s00299-002-0475-7	journal	July 2002
Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein, sHSP17.7 Sato, Yutaka; Yokoya, Sakiko Plant Cell Reports, Vol. 27, Issue 2 https://doi.org/10.1007/s00299-007-0470-0	journal	October 2007
Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge) Sandhu, Sukhpreet; Altpeter, Fredy Plant Cell Reports, Vol. 27, Issue 11 https://doi.org/10.1007/s00299-008-0599-5	journal	August 2008
Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter Wu, Xiaolan; Shiroto, Yoko; Kishitani, Sachie Plant Cell Reports, Vol. 28, Issue 1 https://doi.org/10.1007/s00299-008-0614-x	journal	September 2008
Characterization of Small Heat Shock Proteins Associated with Maize Tolerance to Combined Drought and Heat Stress Hu, Xiuli; Li, Yanhui; Li, Chaohai Journal of Plant Growth Regulation, Vol. 29, Issue 4 https://doi.org/10.1007/s00344-010-9157-9	journal	May 2010
The activities of four constitutively expressed promoters in single-copy transgenic rice plants for two homozygous generations Bang, Seung Woon; Park, Su-Hyun; Kim, Youn Shic Planta, Vol. 241, Issue 6 https://doi.org/10.1007/s00425-015-2278-4	journal	March 2015
Effects of heat shock and salinity on barley growth and stress-related gene transcription Faralli, M.; Lektemur, C.; Rosellini, D. Biologia plantarum, Vol. 59, Issue 3 https://doi.org/10.1007/s10535-015-0518-x	journal	September 2015
Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane Zhao, Yang; Kim, Jae Y.; Karan, Ratna Plant Molecular Biology, Vol. 100, Issue 3 https://doi.org/10.1007/s11103-019-00856-4	journal	March 2019
Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. hybrids) Kim, Jae Yoon; Gallo, Maria; Altpeter, Fredy Plant Cell, Tissue and Organ Culture (PCTOC), Vol. 108, Issue 2 https://doi.org/10.1007/s11240-011-0043-3	journal	September 2011
Expression of selected heat shock proteins after individually applied and combined drought and heat stress Grigorova, Biliana; Vaseva, Irina I.; Demirevska, Klimentina Acta Physiologiae Plantarum, Vol. 33, Issue 5 https://doi.org/10.1007/s11738-011-0733-9	journal	March 2011
Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–lox Mediated Site-Specific Gene Integration in Rice Akbudak, M. Aydın; Srivastava, Vibha Molecular Biotechnology, Vol. 49, Issue 1 https://doi.org/10.1007/s12033-011-9381-y	journal	January 2011
Heat-inducible Cre-lox system for marker excision in transgenic rice Khattri, Abhilasha; Nandy, Soumen; Srivastava, Vibha Journal of Biosciences, Vol. 36, Issue 1 https://doi.org/10.1007/s12038-011-9010-8	journal	March 2011
Importance of heat shock proteins in maize Pegoraro, Camila; Mertz, Liliane Marcia; da Maia, Luciano Carlos Journal of Crop Science and Biotechnology, Vol. 14, Issue 2 https://doi.org/10.1007/s12892-010-0119-3	journal	June 2011
Functional analysis of the temperature-dependent expression of the barley Hvhsp17 gene promoter in monocot and dicot cell systems Raho, G.; Lupotto, E.; Della Torre P., A. Plant Science, Vol. 106, Issue 1 https://doi.org/10.1016/0168-9452(95)04064-2	journal	March 1995
The effect of heat stress and cadmium ions on the expression of a small hsp gene in barley and maize Gullì, M.; Rampino, P.; Lupotto, E. Journal of Cereal Science, Vol. 42, Issue 1 https://doi.org/10.1016/j.jcs.2005.01.006	journal	July 2005
Protein sHSP26 improves chloroplast performance under heat stress by interacting with specific chloroplast proteins in maize (Zea mays) Hu, Xiuli; Yang, Yanfang; Gong, Fangping Journal of Proteomics, Vol. 115 https://doi.org/10.1016/j.jprot.2014.12.009	journal	February 2015
Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template Barone, Pierluigi; Wu, Emily; Lenderts, Brian Molecular Plant, Vol. 13, Issue 8 https://doi.org/10.1016/j.molp.2020.06.008	journal	August 2020
A small heat shock protein CaHsp25.9 positively regulates heat, salt, and drought stress tolerance in pepper (Capsicum annuum L.) Feng, Xiao-Hui; Zhang, Huai-Xia; Ali, Muhammad Plant Physiology and Biochemistry, Vol. 142 https://doi.org/10.1016/j.plaphy.2019.07.001	journal	September 2019
Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response Wang, Wangxia; Vinocur, Basia; Shoseyov, Oded Trends in Plant Science, Vol. 9, Issue 5 https://doi.org/10.1016/j.tplants.2004.03.006	journal	May 2004
A Golden Gate Modular Cloning Toolbox for Plants Engler, Carola; Youles, Mark; Gruetzner, Ramona ACS Synthetic Biology, Vol. 3, Issue 11 https://doi.org/10.1021/sb4001504	journal	February 2014
HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions Huang, Ying; An, Jing; Sircar, Sanchari Nature Communications, Vol. 14, Issue 1 https://doi.org/10.1038/s41467-023-36227-3	journal	January 2023
Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae Haslbeck, Martin; Braun, Nathalie; Stromer, Thusnelda The EMBO Journal, Vol. 23, Issue 3 https://doi.org/10.1038/sj.emboj.7600080	journal	January 2004
Ethanol treatment triggers a heat shock‐like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings Kuo, H. ‐F.; Tsai, Y. ‐F.; Young, L. ‐S. Plant, Cell & Environment, Vol. 23, Issue 10 https://doi.org/10.1046/j.1365-3040.2000.00621.x	journal	October 2000
Tissue‐specific expression of sunflower heat shock proteins in response to water stress Almoguera, Concepción; Coca, María A.; Jordano, Juan The Plant Journal, Vol. 4, Issue 6 https://doi.org/10.1046/j.1365-313X.1993.04060947.x	journal	December 1993
Heat‐inducible expression of FLP gene in maize cells Lyznik, Leszek A.; Hirayama, Lynne; Rao, K. V. The Plant Journal, Vol. 8, Issue 2 https://doi.org/10.1046/j.1365-313X.1995.08020177.x	journal	August 1995
A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state Lee, G. J. The EMBO Journal, Vol. 16, Issue 3 https://doi.org/10.1093/emboj/16.3.659	journal	February 1997
Hsp26: a temperature-regulated chaperone Haslbeck, M. The EMBO Journal, Vol. 18, Issue 23 https://doi.org/10.1093/emboj/18.23.6744	journal	December 1999
Tissue-10Hvhsp17gene promoter in transgenic tobacco plants Raho, Giovanna; Lupotto, Elisabetta; Hartings, Hans Journal of Experimental Botany, Vol. 47, Issue 10 https://doi.org/10.1093/jxb/47.10.1587	journal	January 1996
PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences Lescot, M. Nucleic Acids Research, Vol. 30, Issue 1 https://doi.org/10.1093/nar/30.1.325	journal	January 2002
Rapid isolation of high molecular weight plant DNA Murray, M. G.; Thompson, W. F. Nucleic Acids Research, Vol. 8, Issue 19 https://doi.org/10.1093/nar/8.19.4321	journal	January 1980
PlantPAN3.0: a new and updated resource for reconstructing transcriptional regulatory networks from ChIP-seq experiments in plants Chow, Chi-Nga; Lee, Tzong-Yi; Hung, Yu-Cheng Nucleic Acids Research, Vol. 47, Issue D1 https://doi.org/10.1093/nar/gky1081	journal	November 2018
PlantRegMap: charting functional regulatory maps in plants Tian, Feng; Yang, De-Chang; Meng, Yu-Qi Nucleic Acids Research https://doi.org/10.1093/nar/gkz1020	journal	November 2019
The Expression of Small Heat Shock Proteins in Seeds Responds to Discrete Developmental Signals and Suggests a General Protective Role in Desiccation Tolerance Wehmeyer, Nadja; Vierling, Elizabeth Plant Physiology, Vol. 122, Issue 4 https://doi.org/10.1104/pp.122.4.1099	journal	April 2000
Cloning, Sequence Analysis, and Expression of a cDNA Encoding a Plastid-Localized Heat Shock Protein in Maize Nieto-Sotelo, Jorge; Vierling, Elizabeth; Ho, Tuan-Hua David Plant Physiology, Vol. 93, Issue 4 https://doi.org/10.1104/pp.93.4.1321	journal	August 1990
High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Allen, G. C.; Hall, G.; Michalowski, S. The Plant Cell, Vol. 8, Issue 5 https://doi.org/10.1105/tpc.8.5.899	journal	May 1996
Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems Parajuli, Saroj; Kannan, Baskaran; Karan, Ratna GCB Bioenergy, Vol. 12, Issue 7 https://doi.org/10.1111/gcbb.12684	journal	May 2020
Molecular responses of plants to an increased incidence of heat shock Howarth, C. J. Plant, Cell & Environment, Vol. 14, Issue 8 https://doi.org/10.1111/j.1365-3040.1991.tb01446.x	journal	October 1991
Thirty years of plant transformation technology development Vain, Philippe Plant Biotechnology Journal, Vol. 5, Issue 2 https://doi.org/10.1111/j.1467-7652.2006.00225.x	journal	March 2007
Temporal and spatial control of transgene expression using a heat‐inducible promoter in transgenic wheat Freeman, Jacqueline; Sparks, Caroline A.; West, Jevon Plant Biotechnology Journal, Vol. 9, Issue 7 https://doi.org/10.1111/j.1467-7652.2011.00588.x	journal	January 2011
Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice Palaniswamy, Harunipriya; Syamaladevi, Divya P.; Mohan, Chakravarthi Plant Biotechnology Journal, Vol. 14, Issue 2 https://doi.org/10.1111/pbi.12430	journal	July 2015
Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis Guan, Qingmei; Lu, Xiaoyan; Zeng, Haitao The Plant Journal, Vol. 74, Issue 5 https://doi.org/10.1111/tpj.12169	journal	April 2013
Localization of Small Heat Shock Proteins to the Higher Plant Endomembrane System Helm, Kenneth W.; LaFayette, Peter R.; Nagao, Ronald T. Molecular and Cellular Biology, Vol. 13, Issue 1 https://doi.org/10.1128/mcb.13.1.238-247.1993	journal	January 1993
Regulatory Domains of the Gmhsp17.5-E Heat Shock Promoter of Soybean Czarnecka, Eva; Key, Joe L.; Gurley, William B. Molecular and Cellular Biology, Vol. 9, Issue 8 https://doi.org/10.1128/mcb.9.8.3457-3463.1989	journal	August 1989
Identification, characterization, and analysis of cDNA and genomic sequences encoding two different small heat shock proteins in Hordeum vulgare Marmiroli, Nelson; Pavesi, Angelo; Cola, Gabriella Di Genome, Vol. 36, Issue 6 https://doi.org/10.1139/g93-148	journal	December 1993
The Roles of Heat Shock Proteins in Plants Vierling, E. Annual Review of Plant Physiology and Plant Molecular Biology, Vol. 42, Issue 1 https://doi.org/10.1146/annurev.pp.42.060191.003051	journal	June 1991
Solar Heating (Solarization) of Soil for Control of Soilborne Pests Katan, J. Annual Review of Phytopathology, Vol. 19, Issue 1 https://doi.org/10.1146/annurev.py.19.090181.001235	journal	September 1981
Expression and Promoter Analysis of Six Heat Stress‐Inducible Genes in Rice Rerksiri, Wirat; Zhang, Xianwen; Xiong, Hairong The Scientific World Journal, Vol. 2013, Issue 1 https://doi.org/10.1155/2013/397401	journal	January 2013
A heat-shock inducible system for flexible gene expression in cereals Harrington, Sophie A.; Backhaus, Anna E.; Fox, Samantha Plant Methods, Vol. 16, Issue 1 https://doi.org/10.1186/s13007-020-00677-3	journal	October 2020
Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues Cao, Viet Dang; Luo, Guangbin; Korynta, Shelby Biotechnology for Biofuels and Bioproducts, Vol. 16, Issue 1 https://doi.org/10.1186/s13068-023-02393-1	journal	October 2023
Wheat Chloroplast Targeted sHSP26 Promoter Confers Heat and Abiotic Stress Inducible Expression in Transgenic Arabidopsis Plants Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit PLoS ONE, Vol. 8, Issue 1 https://doi.org/10.1371/journal.pone.0054418	journal	January 2013
Back to the basics: the molecular blueprint of plant heat stress transcription factors Fragkostefanakis, Sotirios; Schleiff, Enrico; Scharf, Klaus-Dieter Biological Chemistry, Vol. 406, Issue 5-7 https://doi.org/10.1515/hsz-2025-0115	journal	April 2025
Identification, Characterization and Expression of Drought Related alpha‐Crystalline Heat Shock Protein Gene (GHSP26) from Desi Cotton Maqbool, Asma; Zahur, Muzna; Irfan, Muhammad Crop Science, Vol. 47, Issue 6 https://doi.org/10.2135/cropsci2007.03.0120	journal	November 2007
Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels Hoang, Nam V.; Furtado, Agnelo; Botha, Frederik C. Frontiers in Bioengineering and Biotechnology, Vol. 3 https://doi.org/10.3389/fbioe.2015.00182	journal	November 2015
High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane Padilla, Carmen S.; Damaj, Mona B.; Yang, Zhong-Nan Frontiers in Bioengineering and Biotechnology, Vol. 8 https://doi.org/10.3389/fbioe.2020.00977	journal	August 2020
Crop Production under Drought and Heat Stress: Plant Responses and Management Options Fahad, Shah; Bajwa, Ali A.; Nazir, Usman Frontiers in Plant Science, Vol. 8 https://doi.org/10.3389/fpls.2017.01147	journal	June 2017
An Efficient Gene Excision System in Maize Wang, Ning; Arling, Maren; Hoerster, George Frontiers in Plant Science, Vol. 11 https://doi.org/10.3389/fpls.2020.01298	journal	September 2020
Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells Sheva, Maor; Hanania, Uri; Ariel, Tami Frontiers in Plant Science, Vol. 11 https://doi.org/10.3389/fpls.2020.607174	journal	November 2020
Transcriptional Regulatory Network of Plant Cadmium Stress Response Li, Yakun; Ding, Lihong; Zhou, Mei International Journal of Molecular Sciences, Vol. 24, Issue 5 https://doi.org/10.3390/ijms24054378	journal	February 2023
Mechanisms of Plant Epigenetic Regulation in Response to Plant Stress: Recent Discoveries and Implications Abdulraheem, Mukhtar Iderawumi; Xiong, Yani; Moshood, Abiodun Yusuff Plants, Vol. 13, Issue 2 https://doi.org/10.3390/plants13020163	journal	January 2024

Similar Records

AAVPG: A vigilant vector where transgene expression is induced by p53

Journal Article · Sat Dec 14 23:00:00 EST 2013 · Virology · OSTI ID:22436664

Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue

Journal Article · Tue Sep 07 00:00:00 EDT 2021 · Biotechnology for Biofuels · OSTI ID:1827191

Related Subjects

GUS activity
heat inducible promoter
in silico analysis of heat shock elements
stem
sugarcane
transgene expression
uidA
vegetative tissue

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

Citation Formats

References (67)

Similar Records

Related Subjects