Activation of PIF4/5 by the transcription factor TCP4 promotes hypocotyl elongation in Arabidopsis

Activation of PIF4/5 by the transcription factor TCP4 promotes hypocotyl elongation in Arabidopsis | 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 Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed.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. 3 March 2025 V1 Latest version Share on Activation of PIF4/5 by the transcription factor TCP4 promotes hypocotyl elongation in Arabidopsis Authors : Yu Zhang 0009-0004-6445-894X [email protected] , Qidong Lei , Caspar Chater 0000-0003-2058-2020 , Yuan Duan 0000-0002-8399-5116 , Yong Yang , and Xu-dong Sun 0000-0002-2434-8031 Authors Info & Affiliations https://doi.org/10.22541/au.174098544.44541101/v1 Published Physiologia Plantarum Version of record Peer review timeline 219 views 138 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Transcription factor TEOSINTE BRANCHED1, CYCLODEA, PROLIFERATING CELL FACTORS 4 (TCP4) plays essential roles in plant development processes.The role of TCP4 in the regulation of hypocotyl growth remains a matter of debate.Our results cast doubt on the previous claims that TCP4 directly activates YUC5 expression to promote hypocotyl elongation, and clearly show that TCP4 promotes hypocotyl elongation dependent on PIF4/5 . Short title: TCP4 targets PIF4/5 to control hypocotyl growth Yu Zhang 1,5 , Qidong Lei 1,2 , Caspar C. C. Chater 3,4 , Yuanwen Duan 1,5 , Yongping Yang 1,5* , Xudong Sun 1,5* 1 Yunnan Key Laboratory of Crop Wild Relatives Omics, The Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China 2 Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China 3 Royal Botanic Gardens, Kew, Richmond, TW9 3AE, United Kingdom 4 Plants, Photosynthesis and Soil, School of Biosciences, University of Sheffield, S10 2TN, United Kingdom 5 University of Chinese Academy of Sciences, Beijing, China *Correspondence: *Correspondence author e-mail: [email protected] ; [email protected] Tel: 86-871-65230873 The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Xudong Sun ( [email protected] ). Dear Editor, Transcription factor TEOSINTE BRANCHED1, CYCLODEA, PROLIFERATING CELL FACTORS 4 (TCP4) plays essential roles in plant development processes, such as leaf morphogenesis (Bresso et al., 2017; Li et al., 2012; Palatnik et al., 2003; Schommer et al., 2008), jasmonic acid biosynthesis (Schommer et al. , 2008), cell proliferation (Schommer et al., 2014), flower development and flowering time (Kubota et al., 2017; Nag et al., 2009), and secondary cell wall synthesis (Sun et al., 2017). Overexpression of miR319-resistant TCP4 results in long hypocotyls, hyponastic cotyledons, and smaller rosette leaves. Jaw - D , a miR319a overexpressing line, has opposite phenotypes (Schommer et al. , 2008; Sun et al. , 2017). We recently identified that a UVR8-TCP4-LOX2 module regulates UV-B tolerance dependent on the JA signaling pathway in Arabidopsis thaliana , and TCP4 regulates UV‐B tolerance independently of ELONGATED HYPOCOTYL 5 (HY5) signaling (Li et al., 2024). HY5 is the central regulator of light signal transduction for hypocotyl growth and anthocyanin homeostasis (Contreras-Avilés et al., 2024). These results also indicated that TCP4 regulates hypocotyl elongation independent of HY5. Challa et al. (2016) reported that TCP4 directly activates YUCCA5 ( YUC5 ) expression to promote Arabidopsis hypocotyl elongation. Although overexpression of YUC5 causes elongated hypocotyls and narrow leaves, the cotyledons are not hyponastic (Woodward et al., 2005). Similarly, hypocotyl lengths did not show obvious differences in yuc5 mutants compared to WT (Müller-Moulé et al., 2016). The role of TCP4, therefore, in the regulation of hypocotyl growth remains a matter of debate. To test the genetic relationship between TCP4 and YUC5 , we crossed TCP4 with yucQ , a yuc3/5/7/8/9 quintuple mutant. Our results showed that whereas the yucQ mutant had a WT-like hypocotyl length, rTCP4-3 x yucQ had a similar phenotype to rTCP4-3 (Figure 1A,B). A previous study showed that YUC3 , YUC5 , YUC7 , YUC8 and YUC9 genes are expressed in seedling roots, and that the loss-of-function yucQ mutant generates a short and agravitropic primary root (Chen et al., 2014). Although overexpression of YUC5 resulted in elongated hypocotyls, the loss-of-function of yuc5 mutant did not reduce hypocotyl length (Müller-Moulé et al. , 2016). In the present study, we found no differences in the relative expression of YUC5 between rTCP4-3 and WT hypocotyls (Figure 1C). Together, these results strongly suggested that YUC5 is not the target gene of TCP4 in hypocotyl elongation processes. To begin to elucidate the candidate target genes for TCP4’s regulation of hypocotyl elongation we undertook comparative transcriptome analysis. A total of 4,316 differentially expressed genes (DEGs) were identified (FDR 0.8), 2,033 genes of which were significantly up-regulated and 2,283 genes of which were significantly down-regulated (Figure 1D, Table S1), and the expression patterns of these DEGs in WT and rTCP4-3 were completely opposite (Figure 1E). Correlation heatmap and Principal Component Analysis (PCA) indicated that rTCP4-3 groups and WT groups had distinct gene expression profiles and our analysis was repeatable and stable (Figure S1). By combining RNA-seq with ChIP-seq data of TCP4 (Dong et al., 2019), an overlap was found for 521 up-regulated and 760 down-regulated DEGs between RNA-seq and ChIP-seq experiments (Figure 1F, Table S2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed enrichment of pathways associated with circadian rhythm, starch and sucrose metabolism, and “Plant hormone signal transduction” (Figure 1G). An expression heatmap identified the key components of “Plant hormone signal transduction” and, coupled with qRT-PCR, revealed that PIF4 transcript levels were strongly up-regulated in the hypocotyls of rTCP4 seedlings (Figure 1H,I). To test the genetic relationship between TCP4 and PIF4 , we obtained rTCP4/pifQ , a pif1/3/4/5 quadruple mutant. The resultant hypocotyl phenotype of rTCP4-3 x pifQ was similar to pifQ , with a length significantly shorter than WT (Figure 1J,K), further confirming that the PIFs may be involved in TCP4-mediated hypocotyl elongation. The ChIP assay revealed that TCP4 could directly bind to the promoter of PIF4/5 (Figure 1L). A previous study had also revealed that TCP4 could bind to PIF4/5 in the genome (Dong et al. , 2019). To support this, we undertook a luciferase assay which revealed that TCP4 could positively regulate the promoter activity of PIF4/5 (Figure 1M). Combining all of the above results, we hypothesize that PIF4/5 is the target gene for TCP4-mediated hypocotyl elongation in Arabidopsis . In conclusion, we propose a working model for a TCP4- PIF4/5 regulatory module that governs hypocotyl elongation (Figure 1N). Our results cast doubt on the previous claims that TCP4 directly activates YUC5 expression to promote hypocotyl elongation, and clearly show that TCP4 promotes hypocotyl elongation dependent on PIF4/5 . Figure legends FIGURE 1 | TCP4- PIF4/5 module regulate hypocotyl elongation in Arabidopsis . (A) Hypocotyl phenotype of the indicated genotypes (7-day-old). Notes rTCP4-3 x yucQ showed a similar phenotype to rTCP4-3 . Bar= 0.5 cm. (B) Average hypocotyl length of the indicated genotypes. N= 8. (C) Relative hypocotyl transcript levels of YUC3 , YUC5 , YUC7 , YUC8 and YUC9 were determined by qRT-PCR of the indicated genotypes (7-day-old). (D) Volcano plot of differently expressed genes in the hypocotyl of rTCP4-3 . (E) Heatmap of significant DEGs between rTCP4-3 and WT. (F) Venn diagram analyses showing the common and exclusive DEGs between RNA-seq and ChIP-seq (Dong et al. , 2019). (G) KEGG pathway annotation for the top 20 pathways number of 1,281 DEGs. (H) Differential expression heat map of 31 DEGs in the “Plant hormone signal transduction” pathway. (I) Relative transcript levels of PIF1 , PIF4 and PIF5 in hypocotyl were determined by qRT-PCR of the indicated genotypes (7-day-old). (J) Hypocotyl phenotype of the indicated genotypes (7-day-old). Notes rTCP4-3 x pifQ showed a similar phenotype to pifQ . Bar=0.5 cm. (K) Average hypocotyl length of the indicated genotypes. N= 8. (L) ChIP assays revealed that TCP4 could directly bind to the promoter of PIF4/5 . (M) Luciferase assays showing that TCP4 can positively regulate the promoter of PIF4/5 . (N) Working model for TCP4‐mediated regulation of hypocotyl elongation through activation of PIF4/5 . Supplemental Data Supplemental information is available in the online version of this article. Figure S1. Correlation heatmap and PCA of RNA-seq data. Table S1. List of DEGs in the hypocotyl ( rTCP4 vs WT). Table S2. List of the overlapping genes between the RNA-seq and ChIP-seq experiments (Dong et al., 2019). Table S3. List of the ChIP-qPCR primer sequence. Author Contributions Y.Z., C.C.C., and X.D.S. wrote the article; Y.Z and Q.D.L collected and processed the data; Y.W.D., Y.P.Y., and X.D.S. designed the research. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (NSFC) (No. 32370405), Natural Science foundation of Yunnan Province (2017FB050). Conflicts of Interest The authors declare that they have no conflict of interest. All authors have reviewed and approved the final version of the manuscript. Data Availability Statement All relevant data are available from the corresponding authors upon request. There are no restrictions on data availability. RNA-seq related data were submitted at: https://ngdc.cncb.ac.cn/gsub/submit/gsa/list/CRA018400. Bresso, E.G., Chorostecki, U., Rodriguez, R.E., Palatnik, J.F., and Schommer, C. (2017) Spatial control of gene expression by miR319-regulated TCP transcription factors in leaf development. Plant Physiol 176:1694–1708. Challa, K.R., Aggarwal, P., and Nath, U. (2016) Activation of YUCCA5 by the transcription factor TCP4 integrates developmental and environmental signals to promote hypocotyl elongation in Arabidopsis. Plant Cell 28: 2117–2130.Chen, Q., Dai, X., De-Paoli, H., Cheng, Y., Takebayashi, Y., Kasahara, H., Kamiya, Y., and Zhao, Y. (2014) Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots. Plant Cell Physiol 55:1072–1079. Contreras-Avilés, W., Heuvelink, E., Marcelis, L.F.M., and Kappers, I.F. (2024) Ménage à trois: light, terpenoids, and quality of plants. Trends Plant Sci 29:572–588. 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Sun, X., Wang, C., Xiang, N., Li, X., Yang, S., Du, J., Yang, Y., and Yang, Y. (2017) Activation of secondary cell wall biosynthesis by miR319-targeted TCP4 transcription factor. Plant Biotechnol J 15:1284–1294. Woodward, C., Bemis, S.M., Hill, E.J., Sawa, S., Koshiba, T., and Torii, K.U. (2005) Interaction of auxin and ERECTA in elaborating Arabidopsis inflorescence architecture revealed by the activation tagging of a new member of the YUCCA family putative flavin monooxygenases. Plant Physiol 139:192–203. Supplementary Material File (figure 1.pptx) Download 5.89 MB Information & Authors Information Version history V1 Version 1 03 March 2025 Peer review timeline Published Physiologia Plantarum Version of Record 14 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords development growth hypocotyl pif4/5 tcp4 yuc5 Authors Affiliations Yu Zhang 0009-0004-6445-894X [email protected] Kunming Institute of Botany Chinese Academy of Sciences View all articles by this author Qidong Lei Kunming Institute of Botany Chinese Academy of Sciences View all articles by this author Caspar Chater 0000-0003-2058-2020 Royal Botanic Gardens Kew View all articles by this author Yuan Duan 0000-0002-8399-5116 Kunming Institute of Botany Chinese Academy of Sciences View all articles by this author Yong Yang Kunming Institute of Botany Chinese Academy of Sciences View all articles by this author Xu-dong Sun 0000-0002-2434-8031 Kunming Institute of Botany Chinese Academy of Sciences View all articles by this author Metrics & Citations Metrics Article Usage 219 views 138 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yu Zhang, Qidong Lei, Caspar Chater, et al. 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