Multiomics analysis reveals that VrKNAT6 regulates pod number through jasmonic acid synthesis

doi:10.1016/j.jia.2025.05.015

Advanced Online Publication | Current Issue | Archive | Adv Search

Multiomics analysis reveals that VrKNAT6 regulates pod number through jasmonic acid synthesis

Shicong Li^1,^2*, Jinyang Liu^1*, Dan Gong^1,², Qiang Yan¹, Yun Lin¹,Jingbin Chen¹, Ranran Wu¹, Xi Zhang¹, Yixiang Pu¹, Somta Prakit^3#, Xin Chen¹^#, Xingxing Yuan¹^#

¹Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China

²College of Life Sciences, Nanjing Agricultural University, Nanjing 210000, China

³Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen 73140, Nakhon Pathom, Thailand

Highlights

1. Multiple GWAS methods were employed to identify the mung bean pod development-related gene VrKNAT6.

2. Metabolomic analysis of VrKNAT6^H1-overexpressing mung bean hairy roots and Arabidopsis thaliana demonstrated that VrKNAT6 regulates pod number per plant by participating in JA synthesis.

3. Bioluminescence and luciferase complementation analyses revealed that VrKNAT6 interacts with VrATH1, indicating its involvement in the JA synthesis pathway.

Abstract
References

Download: PDF in ScienceDirect
Export: BibTeX | EndNote (RIS)

摘要

单株荚果数（PP）与种子产量具有强烈的相关性，鉴定调控PP的基因能够增加绿豆产量，为其分子育种提供极具价值的信息，而在本研究中，利用全基因组关联研究和多组学分析鉴定到了单株荚果数调控基因VrKNAT6。利用EMMAX、CMLM、GEMMA、GLM和3VmrMLM等方法筛选到与PP显著相关位点Chr3-14344673 (P = 3.02E-10~8.80E-07)。在Chr3-14344673位点上下游100 kb的区域发现12个候选基因，其中EVM0027029 (VrKNAT6)与水稻和拟南芥中已知的荚果数发育相关基因同源。VrKNAT6^H1过表达能显著增加转基因拟南芥的莲座叶数、分枝数、PP和千粒重。此外，VrKNAT6^H1在绿豆毛状根和拟南芥中过表达时，VrKNAT6^H1可能通过与VrATH1互作参与茉莉酸（JA）合成，部分解释了VrKNAT6^H1过表达株系与对照之间在分枝数方面的差异。并且，在VrKNAT6^H1过表达株系中，与JA合成相关基因的表达量显著升高。本研究基于多组学分析结果，推测了VrKNAT6^H1的分子调控机制，既VrKNAT6^H1作为JA合成相关基因可用于绿豆高产分子育种。

Abstract

The number of pods per plant (PP) is strongly correlated with seed yield, and identifying genes that regulate PP could enhance the yield of mung bean (Vigna radiata (L.) Wilczek), providing valuable insights for molecular breeding. In this study, VrKNAT6 was identified through genome-wide association and multiomics analyses. Chr3-14344673 (P=3.02E-10~8.80E-07) was found to be significantly associated with PP using EMMAX, CMLM, GEMMA, GLM, and 3VmrMLM. Among the 12 genes located within a 100 kb region near Chr3-14344673 on chromosome 3, EVM0027029 (VrKNAT6) is homologous to known PP development-related genes in Oryza sativa and Arabidopsis thaliana. Overexpression of VrKNAT6^H1significantly increased rosette numbers, branch numbers, PP, and the 1,000-seed weight in transgenic Arabidopsis lines. Furthermore, when overexpressed in mung bean hairy roots and Arabidopsis, VrKNAT6^H1 was found to participate in jasmonic acid (JA) synthesis through physical interaction with VrATH1. This interaction partly explains the differences in branch numbers between VrKNAT6^H1-overexpressing Arabidopsis lines and the control. Additionally, the expression of JA synthetase-related genes was significantly elevated in the positive VrKNAT6^H1 lines. Based on the multiomics analysis results, we propose a molecular regulatory mechanism for VrKNAT6^H1,suggesting that it is a JA synthesis-related gene that could be utilized in mung bean high-yield molecular breeding.

Keywords: genome-wide association study multiomics analysis pods per plant trait VrKNAT6 mung bean JA branch numbers VrATH high yield

Online: 16 May 2025

Fund:

This work was supported by the National Natural Science Foundation of China (32200499), the National Key Research and Development Program of China (2023YFD1202702), the China Agriculture Research System of MOF and MARA-Food Legumes (CARS-08-G15), the Jiangsu Seed Industry Revitalization Project, China (JBGS [2021]004), and the NSRF via the Program Management Unit for Human Resources, Institutional Development, Research and Innovation (B16F640185).

About author: #Correspondence Xingxing Yuan, E-mail: yxx@jaas.ac.cn; Xin Chen, E-mail: cx@jaas.ac.cn; Somta Prakit, E-mail: agrpks@ku.ac.th * These authors contributed equally to this work.

	Service
	E-mail this article
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

Shicong Li, Jinyang Liu, Dan Gong, Qiang Yan, Yun Lin, Jingbin Chen, Ranran Wu, Xi Zhang, Yixiang Pu, Somta Prakit, Xin Chen, Xingxing Yuan. 2025. Multiomics analysis reveals that VrKNAT6 regulates pod number through jasmonic acid synthesis. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.05.015

Alam M K, Islam M M, Salahin N, Hasanuzzaman M. 2014. Effect of tillage practices on soil properties and crop productivity in wheat-mungbean-rice cropping system under subtropical climatic conditions. The Scientific World Journal, 2014, 437283.

Barrett J C, Fry B, Maller J, Daly M J. 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics, 21, 263-265.

Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13, 1194-1202.

Cline M G. 1996. Exogenous auxin effects on lateral bud outgrowth in decapitated shoots. Annals of Botany, 78, 255-266.

Crick J, Corrigan L, Belcram K, Khan M, Dawson J W, Adroher B, Li S, Hepworth S R, Pautot V. 2022. Floral organ abscission in Arabidopsis requires the combined activities of three TALE homeodomain transcription factors. Journal of Experimental Botany, 73, 6150-6169.

Delfini J, Moda-Cirino V, Dos Santos Neto J, Zeffa D M, Nogueira A F, Ribeiro L A B, Ruas P M, Gepts P, Goncalves L S A. 2021. Genome-wide association study identifies genomic regions for important morpho-agronomic traits in mesoamerican common bean. Frontiers in Plant Science, 12, 748829.

Di G, Elisabetta, Maria A I, Giovanna F. 2013. TALE and shape: How to make a leaf different. Plants, 2, 317-342.

Ellis C, Karafyllidis I, Wasternack C, Turner J G. 2002. The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. The Plant Cell, 14, 1557-1566.

Fang Y, Zhang X, Liu H, Wu J, Qi F, Sun Z, Zheng Z, Dong W, Huang B. 2023. Identification of quantitative trait loci and development of diagnostic markers for growth habit traits in peanut (Arachis hypogaea L.). Theoretical and Applied Genetics, 136, 105.

Furuta Y, Yamamoto H, Hirakawa T, Uemura A, Pelayo M A, Iimura H, Katagiri N, Takeda-Kamiya N, Kumaishi K, Shirakawa M, Ishiguro S, Ichihashi Y, Suzuki T, Goh T, Toyooka K, Ito T, Yamaguchi N. 2024. Petal abscission is promoted by jasmonic acid-induced autophagy at Arabidopsis petal bases. Nature Communications, 15, 1098.

Hamwieh A, Imtiaz M, Malhotra R S. 2013. Multi-environment QTL analyses for drought-related traits in a recombinant inbred population of chickpea (Cicer arietinum L.). Theoretical and Applied Genetics, 126, 1025-1038.

Heinrich M, Hettenhausen C, Lange T, Wunsche H, Fang J, Baldwin I T, Wu J. 2013. High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems. The Plant Journal, 73, 591-606.

Heitz T, Bergey D R, Ryan C A. 1997. A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiology, 114, 1085-1093.

Horn S, Figl A, Rachakonda P S, Fischer C, Sucker A, Gast A, Kadel S, Moll I, Nagore E, Hemminki K, Schadendorf D, Kumar R. 2013. TERT promoter mutations in familial and sporadic melanoma. Science, 339, 959-961.

Ikeda T, Ohnishi S, Senda M, Miyoshi T, Ishimoto M, Kitamura K, Funatsuki H. 2009. A novel major quantitative trait locus controlling seed development at low temperature in soybean (Glycine max). Theoretical and Applied Genetics, 118, 1477-1488.

Jiang H L, Hong J, Jiang Y T, Yu S X, Zhang Y J, Shi J X, Lin W H. 2020. Genome-wide association analysis identifies candidate genes regulating seed number per silique in Arabidopsis thaliana. Plants (Basel), 9 .

Jin G, Deng Z, Wang H, Zhang Y, Fu R. 2024. EpMYB2 positively regulates chicoric acid biosynthesis by activating both primary and specialized metabolic genes in purple coneflower. The Plant journal, 119, 252-265.

Kang H M, Sul J H, Service S K, Zaitlen N A, Kong S Y, Freimer N B, Sabatti C, Eskin E. 2010. Variance component model to account for sample structure in genome-wide association studies. Nature Genetics, 42, 348-354.

Kang Y J, Kim S K, Kim M Y, Lestari P, Kim K H, Ha B K, Jun T H, Hwang W J, Lee T, Lee J, Shim S, Yoon M Y, Jang Y E, Han K S, Taeprayoon P, Yoon N, Somta P, Tanya P, Kim K S, Gwag J G, et al. 2014. Genome sequence of mungbean and insights into evolution within Vigna species. Nature Communications, 5, 5443.

Kereszt A, Li D, Indrasumunar A, Nguyen C D, Nontachaiyapoom S, Kinkema M, Gresshoff P M. 2007. Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nature Protocols, 2, 948-952.

Khan H A, Siddique K H, Munir R, Colmer T D. 2015. Salt sensitivity in chickpea: Growth, photosynthesis, seed yield components and tissue ion regulation in contrasting genotypes. Journal of Plant Physiology, 182, 1-12.

Khan M, Ragni L, Tabb P, Salasini B C, Chatfield S, Datla R, Lock J, Kuai X, Despres C, Proveniers M, Yongguo C, Xiang D, Morin H, Rulliere J P, Citerne S, Hepworth S R, Pautot V. 2015. Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis. Plant Physiology, 169, 2166-2186.

Li M, Zhang Y W, Xiang Y, Liu M H, Zhang Y M. 2022. IIIVmrMLM: The R and C++ tools associated with 3VmrMLM, a comprehensive GWAS method for dissecting quantitative traits. Molecular Plant, 15, 1251-1253.

Li Y, Pi L, Huang H, Xu L. 2012. ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture. Journal of Experimental Botany, 63, 1423-1433.

Liao L Y, He Z Q, Zhang L. 2023. Cloning and functional analysis of the VfRR17 gene from tung tree (Vernicia fordii). Plants (Basel), 12 .

Liu J, Lin Y, Chen J, Yan Q, Xue C, Wu R, Chen X, Yuan X. 2022a. Genome-wide association studies provide genetic insights into natural variation of seed-size-related traits in mungbean. Frontiers in Plant Science, 13, 997988.

Liu J, Xue C, Lin Y, Yan Q, Chen J, Wu R, Zhang X, Chen X, Yuan X. 2022b. Genetic analysis and identification of VrFRO8, a salt tolerance-related gene in mungbean. Gene, 836, 146658.

Liu J Y, Li P, Zhang Y W, Zuo J F, Li G, Han X, Dunwell J M, Zhang Y M. 2020. Three-dimensional genetic networks among seed oil-related traits, metabolites and genes reveal the genetic foundations of oil synthesis in soybean. The Plant Journal, 103, 1103-1124.

Long J, Barton M K. 2000. Initiation of axillary and floral meristems in Arabidopsis. Developmental Biology, 218, 341-353.

Lu K, Peng L, Zhang C, Lu J, Yang B, Xiao Z, Liang Y, Xu X, Qu C, Zhang K, Liu L, Zhu Q, Fu M, Yuan X, Li J. 2017. Genome-wide association and transcriptome analyses reveal candidate genes underlying yield-determining traits in Brassica napus. Frontiers in Plant Science, 8, 206.

Ma X, Ying P, He Z, Wu H, Li J, Zhao M. 2021. The LcKNAT1-LcEIL2/3 regulatory module is involved in fruitlet abscission in litchi. Frontiers in Plant Science, 12, 802016.

Mahajan A S, Kondhare K R, Rajabhoj M P, Kumar A, Ghate T, Ravindran N, Habib F, Siddappa S, Banerjee A K. 2016. Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato. Journal of Experimental Botany, 67, 4255-4272.

Müssig C, Biesgen C, Lisso J, Uwer U, Weiler E W, Altmann T. 2000. A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between Brassinosteroid-action and Jasmonic-acid synthesis. Journal of Plant Physiology, 157, 143-152.

Nguyen T H, Goossens A, Lacchini E. 2022. Jasmonate: A hormone of primary importance for plant metabolism. Current Opinion in Plant Biology, 67, 102197.

Nidhi S, Preciado J, Tie L. 2021. Knox homologs shoot meristemless (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during shoot meristem development in Arabidopsis thaliana. Molecular Biology Reports, 48, 6291-6302.

Ning H, Yuan J, Dong Q, Li W, Xue H, Wang Y, Tian Y, Li W X. 2018. Identification of QTLs related to the vertical distribution and seed-set of pod number in soybean [Glycine max (L.) Merri]. PLoS ONE, 13, e0195830.

Nooden L D, Nooden S M. 1985. Effects of morphactin and other auxin transport inhibitors on soybean senescence and pod development. Plant Physiology, 78, 263-266.

Nowak J S, Bolduc N, Dengler N G, Posluszny U. 2011. Compound leaf development in the palm Chamaedorea elegans is KNOX-independent. American Journal of Botany, 98, 1575-1582.

Nygren H, Seppänen-Laakso T, Castillo S, Hyötyläinen T, Orešič M. 2011. Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics for studies of body fluids and tissues. Molecular Biology Reports, 708, 247-257.

Pongpanich M, Sullivan P F, Tzeng J Y. 2010. A quality control algorithm for filtering SNPs in genome-wide association studies. Bioinformatics, 26, 1731-1737.

Qi Z, Song J, Zhang K, Liu S, Tian X, Wang Y, Fang Y, Li X, Wang J, Yang C, Jiang S, Sun X, Tian Z, Li W, Ning H. 2020. Identification of QTNs controlling 100-seed weight in soybean using multilocus genome-wide association studies. Frontiers in Genetics, 11, 689.

Ragni L, Belles-Boix E, Gunl M, Pautot V. 2008. Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis inflorescences. The Plant Cell, 20, 888-900.

Sasaki Y, Asamizu E, Shibata D, Nakamura Y, Kaneko T, Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T, Shimada H, Takamiya K, Ohta H, Tabata S. 2001. Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways. DNA Research, 8, 153-161.

Scofield S, Dewitte W, Murray J A. 2008. A model for Arabidopsis class-1 KNOX gene function. Plant Signaling & Behavior, 3, 257-259.

Shi J, Zhan J, Yang Y, Ye J, Huang S, Li R, Wang X, Liu G, Wang H. 2015. Linkage and regional association analysis reveal two new tightly-linked major-QTLs for pod number and seed number per pod in rapeseed (Brassica napus L.). Scientific Reports, 5, 14481.

Shin J H, Blay S, McNeney B, Graham J. 2006. LDheatmap: An R function for graphical display of pairwise linkage disequilibria between single nucleotide polymorphisms. Journal of Statistical Software, 16, 1 - 9.

Somta P, Laosatit K, Yuan X, Chen X. 2022. Thirty years of mungbean genome research: Where do we stand and what have we learned? Frontiers in Plant Science, 13, 944721.

Sun D, Li W, Zhang Z, Chen Q, Ning H, Qiu L, Sun G. 2006. Quantitative trait loci analysis for the developmental behavior of Soybean (Glycine max L. Merr.). Theoretical and Applied Genetics, 112, 665-673.

Taniguchi T, Murayama N, Hasegawa M, Nakagawa A C S, Tanaka S, Zheng S H, Hamaoka N, Iwaya-Inoue M, Ishibashi Y. 2018. Vegetative growth after flowering through gibberellin biosynthesis regulates pod setting rate in soybean (Glycine max (L.) Merr.). Plant Signaling & Behavior, 13, e1473668.

Wang G, Zhang X, Huang W, Xu P, Lv Z, Zhao L, Wen J, Yi B, Ma C, Tu J, Fu T, Shen J. 2021. Increased seed number per silique in Brassica juncea by deleting cis-regulatory region affecting BjCLV1 expression in carpel margin meristem. Plant Biotechnology Journal, 19, 2333-2348.

Wang J, Yu J, Lipka A E, Zhang Z. 2022. Interpretation of manhattan plots and other outputs of genome-wide association studies. Methods in Molecular Biology, 2481, 63-80.

Wang L, Qian Y, Wu L, Wei K, Wang L. 2024. The MADS-box transcription factor CsAGL9 plays essential roles in seed setting in Camellia sinensis. Plant Physiology and Biochemistry, 207, 108301.

Xiong S S, Guo D D, Wan Z, Quan L, Lu W T, Xue Y, Liu B, Zhai H. 2023. Regulation of soybean stem growth habit: A ten-year progress report. The Crop Journal, 11, 1642-1648.

Yasmeen E, Wang J, Riaz M, Zhang L, Zuo K. 2023. Designing artificial synthetic promoters for accurate, smart, and versatile gene expression in plants. Plant Communications, 4, 100558.

Ye J, Yang Y, Chen B, Shi J, Luo M, Zhan J, Wang X, Liu G, Wang H. 2017. An integrated analysis of QTL mapping and RNA sequencing provides further insights and promising candidates for pod number variation in rapeseed (Brassica napus L.). BMC Genomics, 18, 71.

Yu H, Bhat J A, Li C, Zhao B, Bu M, Zhang Z, Guo T, Feng X. 2024. Identification of superior and rare haplotypes to optimize branch number in soybean. Theoretical and Applied Genetics, 137, 93.

Yue L, Li X, Fang C, Chen L, Yang H, Yang J, Chen Z, Nan H, Chen L, Zhang Y, Li H, Hou X, Dong Z, Weller J L, Abe J, Liu B, Kong F. 2021. FT5a interferes with the Dt1-AP1 feedback loop to control flowering time and shoot determinacy in soybean. Journal of Integrative Plant Biology, 63, 1004-1020.

Zhang L, Liu J Y, Gu H, Du Y, Zuo J F, Zhang Z, Zhang M, Li P, Dunwell J M, Cao Y, Zhang Z, Zhang Y M. 2018. Bradyrhizobium diazoefficiens USDA 110- Glycine max interactome provides candidate proteins associated with symbiosis. Journal of Proteome Research, 17, 3061-3074.

Zhang W J, Zhai L M, Yu H X, Peng J, Wang S S, Zhang X S, Su Y H, Tang L P. 2020. The BIG gene controls size of shoot apical meristems in Arabidopsis thaliana. Plant Cell Reports, 39, 543-552.

Zhang Z, Ersoz E, Lai C Q, Todhunter R J, Tiwari H K, Gore M A, Bradbury P J, Yu J, Arnett D K, Ordovas J M, Buckler E S. 2010. Mixed linear model approach adapted for genome-wide association studies. Nature Genetics, 42, 355-360.

Zhao B, Liu Q, Wang B, Yuan F. 2021. Roles of phytohormones and their signaling pathways in leaf development and stress responses. Journal of Agricultural and Food Chemistry, 69, 3566-3584.

Zhao M, Li C, Ma X, Xia R, Chen J, Liu X, Ying P, Peng M, Wang J, Shi C L, Li J. 2020. KNOX protein KNAT1 regulates fruitlet abscission in litchi by repressing ethylene biosynthetic genes. Journal of Experimental Botany, 71, 4069-4082.

Zhao M, Yang S, Chen C Y, Li C, Shan W, Lu W, Cui Y, Liu X, Wu K. 2015. Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture. PLoS Genetics, 11, e1005125.

Zhou X, Stephens M. 2012. Genome-wide efficient mixed-model analysis for association studies. Nature Genetics, 44, 821-824.

No related articles found!

No Suggested Reading articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed

Cite this article:

About JIA

Editorial board

For authors