MYB Transcription factor GhMYB4 coordinates transcriptional networks to confer drought resilience in Gossypium hirsutum L.

MYB Transcription factor GhMYB4 coordinates transcriptional networks to confer drought resilience in Gossypium hirsutum L. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 April 2025 V1 Latest version Share on MYB Transcription factor GhMYB4 coordinates transcriptional networks to confer drought resilience in Gossypium hirsutum L. Authors : Yi Wang 0009-0001-1019-2814 , Yuehua Yu , and Zhiyong Ni 0000-0002-6916-069X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174418072.20432747/v1 168 views 101 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract As a globally strategic fiber crop, cotton (Gossypium hirsutum L.) faces escalating yield constraints from abiotic stresses, particularly drought [(Zhu, 2016)](#ref-0001). Therefore, finding genes that increase cotton drought tolerance, while cultivating genetically modified cotton with enhanced drought tolerance, is currently one of the important breeding goals for breeders [(Genc, et al., 2019)](#ref-0002). This study functionally characterizes an R2R3-MYB transcriptional regulator, GhMYB4, that orchestrates molecular responses to water-deficit conditions. –‌ MYB Transcription factor GhMYB4 coordinates transcriptional networks to confer drought resilience in Gossypium hirsutum L. Yi Wang a , Yuehua Yu b, * and Zhiyong Ni a, * a Key Laboratory of Ecological Adaptation and Evolution of Extreme Environment in Xinjiang, College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, China; b College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China *Corresponding author: Zhiyong Ni, E-mail: [email protected] Yuehua Yu, E-mail: [email protected] Keywords: cotton, MYB, transcriptional factor, drought. As a globally strategic fiber crop, cotton ( Gossypium hirsutum L.) faces escalating yield constraints from abiotic stresses, particularly drought (Zhu, 2016). Therefore, finding genes that increase cotton drought tolerance, while cultivating genetically modified cotton with enhanced drought tolerance, is currently one of the important breeding goals for breeders (Genc , et al. , 2019). This study functionally characterizes an R2R3-MYB transcriptional regulator, GhMYB4, that orchestrates molecular responses to water-deficit conditions. In this study, we cloned an R2R3-MYB transcription factor, GhMYB4, which contains dual evolutionarily conserved SANT motifs (aa 13-63; aa 66-114) (Figure 1a). During the seedling stage, GhMYB4 expression exhibited maximal induction (3.5-fold) following 2 h ABA exposuret and exhibited maximal induction (2.7-fold) following 4 h PEG exposure (Figure 1b). The protein encoded by the GhMYB4 gene is localized within the nucleus (Figure 1c) and exhibits no transcriptional activation activity (Figure 1d). To investigate the role of the GhMYB4 gene in cotton’s drought stress response, we overexpressed and miRNA-silenced GhMYB4 in upland cotton ( Gossypium hirsutum L.) variety CQJ-5 (Figure 1s). In the absence of treatment, no significant phenotypic differences were observed among CQJ-5, GhMYB4 -OE, and GhMYB4 -miRNA lines (Figure 1e, h). After 10 days of drought stress, CQJ-5 exhibited a more severe degree of wilting compared to GhMYB4 -OE (Figure 1e), while GhMYB4 -miRNA displayed an even more pronounced wilting than CQJ-5 (Figure 1e). Notably, the survival rate of GhMYB4 -OE was the highest, ranging from 80% to 96.7%, whereas CQJ-5’s survival rate was 72.2%. GhMYB4 -miRNA had the lowest survival rate, spanning from 5.9% to 22.5% (Figure 1f). Consistent with these findings, GhMYB4 -OE maintained a higher chlorophyll content than CQJ-5 both before and after drought stress, whereas GhMYB4 -miRNA had a lower chlorophyll content than CQJ-5 in all instances (Figure 1g). These results indicate that GhMYB4 plays a crucial role in cotton’s drought tolerance. To elucidate how GhMYB4 regulates drought tolerance in cotton, we conducted a preliminary screening of GhMYB4’s interacting proteins using yeast two-hybrid libraries. Our results showed that GhMYB4 could interact with GhMYB6, GhLBD42, and GhWRKY44 (Figure 1h). We further validated this interaction through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) experiments (Figure 1i-l). GhMYB6, GhLBD42, and GhWRKY44 were all differentially expressed in response to drought and ABA treatments (Figure 2s). Silencing these genes using virus-induced gene silencing (VIGS) in cotton reduced their drought tolerance, respectively (Figure 3s-5s). These findings suggest that GhMYB4 regulates cotton’s adaptation to drought stress by interacting with GhMYB6, GhLBD42, and GhWRKY44. To identify the downstream genes regulated by GhMYB4 , we combined DNA affinity purification sequencing (DAP-seq) and RNA sequencing (RNA-seq) of GhMYB4 -OE and GhMYB4 -miRNA lines. From this analysis, we identified a total of 170 target genes that were directly bound and regulated by GhMYB4 (Figure 1m). Among these, 21 genes were up-regulated and 43 genes were down-regulated in GhMYB4 -OE (Figure 1m). In contrast, 64 genes were up-regulated and 42 genes were down-regulated in GhMYB4 -miRNA (Figure 1m). By integrating RNA-seq and DAP-seq data, we screened a candidate downstream gene, GhERF109 , whose featuring canonical MYB-binding MBS elements (CAGTTG) and a not-yet-known not-yet-known not-yet-known unknown Figure 1 GhMYB4 is regulating drought tolerance in cotton. (a) Conserved structural domains of GhMYB4-encoded protein. (b) Transcript levels of GhMYB4 in ABA and PEG. (c) Subcellular localization of GhMYB4 protein. (d) GhMYB4 protein self-activation activity. (e) Phenotypes of CQJ-5, GhMYB4 -OE and GhMYB4 -miRNA under normal and drought stress. (f) Survival rate under drought stress. (g) Chlorophyll content under normal and drought stress. (h-l) Yeast two-hybrid assay (h), BiFC (i) and Co-IP (j-l) show that GhMYB4 interacts with GhMYB6, GhLBD42 and GhWRKY44. (m) Combined analysis of DAP-seq and RNA-seq. (n-p) Yeast single-hybrid assay (n), LUC (o-p), and EMSA (q) demonstrated that GhMYB4 binds to the MBS element in the GhERF109 promoter. (r-t) Yeast single-hybrid assay (r), LUC (s), and EMSA (t) demonstrated that GhMYB4 binds to the KTTTGTTT element in the GhERF109 promoter.(u) Model where GhMYB4 transcription factors are activated under drought stress conditions. novel KTTTGTTT motif. GhERF109 ’s expression was up-regulated in GhMYB4 -OE (Figure 5s). Yeast one-hybrid, dual-luciferase (LUC) assay, and electrophoretic mobility shift assay (EMSA) experiments demonstrated that GhMYB4 could bind to not only the two MBS elements in the GhERF109 promoter (Figure 1n-q) but also to the KTTTGTTT neo-element predicted by DAP-seq (Figure 1r-t). Silencing GhERF109 using VIGS reduced drought tolerance in cotton (Figure 6s). In summary, we have identified an R2R3-MYB transcription factor, GhMYB4, that positively regulates drought tolerance in cotton. We propose a model where the GhMYB4 transcription factor is activated under drought stress conditions (Figure 1u). Upon drought stress initiation, GhMYB4 interacts with GhMYB6, GhLBD42, and GhWRKY44. Simultaneously, GhMYB4 binds to the MBS element and the KTTTGTTT element in the GhERF109 promoter to co-regulate drought tolerance in cotton. This work establishes GhMYB4 as a central hub in drought-responsive signaling cascades, mediating both protein interaction networks (GhMYB6/GhLBD42/GhWRKY44) and hierarchical transcriptional control of effector genes like GhERF109. The discovered KTTTGTTT element expands the known repertoire of MYB-associated cis-motifs in plants. Acknowledgements We thank Wuhan BlOVIVO, China for providing G Pro. This work was supported by the Xinjiang Uygur Autonomous Region Major Science and Technology Project (2024A02002-3), the Xinjiang Uygur Autonomous Region “Tianchi Talents” Introduction Programme and the Xinjiang Uygur Autonomous Region “Tianshan Talents” Cultivation Programme (2022TSYCCX0080). not-yet-known not-yet-known not-yet-known unknown Conflict of interest The authors declare no conflict of interest. Author contributions Z.N., and Y.Y. conceived and designed the experiments. Y.W. and Z.N., performed the experiments. Z.N., Y.Y., and Y.W. wrote the manuscript. not-yet-known not-yet-known not-yet-known unknown References Zhu, J.K. (2016) Abiotic stress signaling and responses in plants. Cell, 167, 313-324.Genc, Y., Taylor, J., Lyons, G., Li, Y., Cheong, J., Appelbee, M., Oldach, K. et al. (2019) Bread wheat with high salinity and sodicity tolerance. Front. Plant Sci. 10, 1280.Supporting information Figure S1 GhMYB4 transcript levels in overexpressed and miRNA silenced lines Figure S2 Transcript levels of GhMYB6, GhLBD42 and GhWRKY44 genes under ABA and drought stress Figure S3 VIGS GhMYB6 reduces drought-resistant in cotton Figure S4 VIGS GhLBD42 reduces drought-resistant in cotton Figure S5 VIGS GhWRKY44 reduces drought-resistant in cotton Figure S6 VIGS GhERF109 reduces drought-resistant in cotton Information & Authors Information Version history V1 Version 1 09 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cotton drought myb transcriptional factor water relations Authors Affiliations Yi Wang 0009-0001-1019-2814 Xinjiang Agricultural University View all articles by this author Yuehua Yu Xinjiang Agricultural University View all articles by this author Zhiyong Ni 0000-0002-6916-069X [email protected] Xinjiang Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 168 views 101 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yi Wang, Yuehua Yu, Zhiyong Ni. MYB Transcription factor GhMYB4 coordinates transcriptional networks to confer drought resilience in Gossypium hirsutum L.. Authorea . 09 April 2025. DOI: https://doi.org/10.22541/au.174418072.20432747/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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