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Journal of Integrative Agriculture  2026, Vol. 25 Issue (7): 2847-2858    DOI: 10.1016/j.jia.2025.11.012
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Functional analysis of CsAGL6 in flower development and pigmentation in cucumber (Cucumis sativus L.)

Li Qin1, 3*, Zheyuan Liu1, 3*, Shuai Li1, 2, 4, Guanghua Cai1, 2, Jie Wang1, Xueyong Yang1, Jinjing Sun1#

1 State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China

2 Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China

3 National Nanfan Research Institute (Sanya), Chinese Academy of Agricultural Sciences, Sanya 572024, China

4 Shandong Key Laboratory of Bulk Open-field Vegetable Breeding, Key Laboratory of Huang Huai Protected Horticulture Engineering of Ministry of Agriculture and Rural Affairs, Institute of Vegetables, Shandong Academy of Agricultural Sciences, Jinan 250100, China

 Highlights 
CsAGL6 participates in sepal and petal development by regulating CAL and SEP4 in cucumber.
CsAGL6 affects petal pigmentation by modulating genes involved in chlorophyll biosynthesis and chloroplast development in cucumber.
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摘要  

黄瓜(Cucumis sativus L.)作为全球重要的蔬菜作物,其产量和品质与花发育密切相关。AGAMOUS-LIKE 6(AGL6)作为古老的MADS-box转录因子家族成员,在花发育过程中具有重要调控作用,但其在黄瓜中的同源基因功能尚未明确。本研究证实CsAGL6主要在花器官中表达,在花发育早期各花器官原基中均呈现高表达水平。在花发育的各个时期,Csagl6突变体的花瓣颜色都较野生型更绿,花瓣总叶绿素含量显著增加;同时Csagl6突变体表现出花瓣形态异常(包括大小和形态改变)及偶尔的萼片增大且叶片化表型。分子机制分析表明,Csagl6突变体中A类基因CAULIFLOWER (CAL)和E类基因SEPALLATA 4 (SEP4)显著下调,而叶绿素合成基因Early Light-Induced Protein 1 (ELIP1)及数个定位于叶绿体的应激反应相关基因显著上调。本研究为解析CsAGL6调控黄瓜萼片和花瓣发育的分子机制提供了新依据,并为理解黄瓜属植物花色素形成及器官形态建成的遗传调控提供了新思路。



Abstract  

Cucumber (Cucumis sativus L.) is a major vegetable crop worldwide, and its yield and quality are closely linked to flower development.  AGAMOUS-LIKE 6 (AGL6), a member of the ancient MADS-box transcription factor family, plays a crucial role in flower development.  However, the specific functions of its homolog in cucumber remain poorly understood.  In this study, we demonstrate that CsAGL6 is predominantly expressed in flowers, with high expression levels observed in all floral organ primordia during the early stages of floral development.  The petals of Csagl6 mutants exhibit a greener color compared to wild-type plants, along with a significant increase in total chlorophyll content.  Additionally, the mutants show abnormal petal morphology, including changes in size and shape, as well as enlarged sepals resembling leaves occasionally.  Molecular analysis reveals that CAULIFLOWER (CAL) and the E-class gene SEPALLATA 4 (SEP4) are significantly downregulated in the mutants, while the chlorophyll synthesis gene Early Light-Induced Protein 1 (ELIP1) and several stress-related genes in the chloroplasts are dramatically upregulated.  Our findings provide novel insights into the functional role of CsAGL6 in regulating sepal and petal development, and offer a potential avenue for understanding the genetic control of flower pigmentation and organ morphology in Cucumis species.

Keywords:  cucumber       flower development       CsAGL6  
Received: 28 March 2025   Accepted: 23 September 2025 Online: 13 November 2025  
Fund: 

This work was supported by funding from the National Natural Science Foundation of China (32222075 and 32472768) and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2024-IVF).

About author:  Li Qin, E-mail: qinliqinfen@163.com; Zheyuan Liu, E-mail: hnlzy1028@163.com; #Correspondence Jinjing Sun, E-mail: sunjinjing@caas.cn * These authors contributed equally to this study.

Cite this article: 

Li Qin, Zheyuan Liu, Shuai Li, Guanghua Cai, Jie Wang, Xueyong Yang, Jinjing Sun. 2026. Functional analysis of CsAGL6 in flower development and pigmentation in cucumber (Cucumis sativus L.). Journal of Integrative Agriculture, 25(7): 2847-2858.

Adamiec M, Dobrogojski J, Wojtyla Ł, Luciński R. 2022. Stress-related expression of the chloroplast EGY3 pseudoprotease and its possible impact on chloroplasts’ proteome composition. Frontiers in Plant Science13, 965143.

Adamiec M, Misztal L, Kasprowicz-Maluśki A, Luciński R. 2020. EGY3: Homologue of S2P protease located in chloroplasts. Plant Biology, 22, 735–743.

Angenent G C, Colombo L. 1996. Molecular control of ovule development. Trends in Plant Science1, 228–232.

Apuya N R, Yadegari R, Fischer R L, Harada J J, Zimmerman J L, Goldberg R B. 2001. The Arabidopsis embryo mutant schlepperless has a defect in the chaperonin-60α gene. Plant Physiology126, 717–730.

Becker A, Theissen G. 2003. The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Molecular Phylogenetics and Evolution29, 464–489.

Bowman J L, Smyth D R, Meyerowitz E M. 2012. The ABC model of flower development: Then and now. Development139, 4095–4098.

Casazza A P, Rossini S, Rosso M G, Soave C. 2005. Mutational and expression analysis of ELIP1 and ELIP2 in Arabidopsis thalianaPlant Molecular Biology58, 41–51.

Causier B, Schwarz-Sommer Z, Davies B. 2010. Floral organ identity: 20 years of ABCs. Seminars in Cell & Development Biology21, 73–79.

Coen E S, Meyerowitz E M. 1991. The war of the whorls: Genetic interactions controlling flower development. Nature353, 31–37.

Cui L, Zheng F Y, Zhang C H, Gao S N, Ye J, Zhang Y Y, Wang T T, Hong Z L, Ye Z B, Zhang J H. 2025. The CONSTANS-LIKE SlCOL1 in tomato regulates the fruit chlorophyll content by stabilizing the GOLDEN2-LIKE protein. Journal of Integrative Agriculture24, 536–545.

Dickson R, Weiss C, Howard R J, Alldrick S P, Ellis R J, Lorimer G, Azem A, Viitanen P V. 2000. Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding. Journal of Biological Chemistry275, 11829–11835.

Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky M F. 2004. The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology14, 1935–1940.

Dreni L, Zhang D. 2016. Flower development: The evolutionary history and functions of the AGL6 subfamily MADS-box genes. Journal of Experiment Botany67, 1625–1638.

Duan Y, Xing Z, Diao Z, Xu W, Li S, Du X, Wu G, Wang C, Lan T, Meng Z, Liu H, Wang F, Wu W, Xue Y. 2012. Characterization of Osmads6-5, a null allele, reveals that OsMADS6 is a critical regulator for early flower development in rice (Oryza sativa L.). Plant Molecular Biology80, 429–442.

Elliot M M, David R S, John L B. 1989. Abnormal flowers and pattern formation in floral development. Development106, 209–217.

Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G, Yanofsky M F, Kater M M, Colombo L. 2003. MADS-box protein complexes control carpel and ovule development in Arabidopsis. The Plant Cell15, 2603–2611.

Hileman L C, Sundstrom J F, Litt A, Chen M, Shumba T, Irish V F. 2006. Molecular and phylogenetic analyses of the MADS-box gene family in tomato. Molecular Biology and Evolution23, 2245–2258.

Hsu H F, Chen W H, Shen Y H, Hsu W H, Mao W T, Yang C H. 2021. Multifunctional evolution of B and AGL6 MADS box genes in orchids. Nature Communications12, 902.

Hsu W H, Yeh T J, Huang K Y, Li J Y, Chen H Y, Yang C H. 2014. AGAMOUS-LIKE13, a putative ancestor for the E functional genes, specifies male and female gametophyte morphogenesis. The Plant Journal77, 1–15.

Huang X, Effgen S, Meyer R C, Theres K, Koornneef M. 2012. Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in ArabidopsisThe Plant Cell24, 2364–2379.

Irish V F. 2010. The flowering of Arabidopsis flower development. The Plant Journal61, 1014–1028.

Klap C, Yeshayahou E, Bolger A M, Arazi T, Gupta S K, Shabtai S, Usadel B, Salts Y, Barg R. 2017. Tomato facultative parthenocarpy results from SlAGAMOUS-LIKE 6 loss of function. Plant Biotechnology Journal15, 634–647.

Kong X, Wang F, Geng S, Guan J, Tao S, Jia M, Sun G, Wang Z, Wang K, Ye X, Ma J, Liu D, Wei Y, Zheng Y, Fu X, Mao L, Lan X, Li A. 2022. The wheat AGL6-like MADS-box gene is a master regulator for floral organ identity and a target for spikelet meristem development manipulation. Plant Biotechnology Journal20, 75–88.

Koo S C, Bracko O, Park M S, Schwab R, Chun H J, Park K M, Seo J S, Grbic V, Balasubramanian S, Schmid M, Godard F, Yun D J, Lee S Y, Cho M J, Weigel D, Kim M C. 2010. Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6The Plant Journal62, 807–816.

Kuo W Y, Huang C H, Liu A C, Cheng C P, Li S H, Chang W C, Weiss C, Azem A, Jinn T L. 2013. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts. New Phytologist197, 99–110.

Latijnhouwers M, Xu X M, Møller S G. 2010. Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development. Planta232, 567–578.

Lee K, Yoon H, Seo P J. 2024. The AGL6-ELF3-FT circuit controls flowering time in ArabidopsisPlant Signaling & Behavior19, 2358684.

Li B J, Zheng B Q, Wang J Y, Tsai W C, Lu H C, Zou L H, Wan X, Zhang D Y, Qiao H J, Liu Z J, Wang Y. 2020. New insight into the molecular mechanism of colour differentiation among floral segments in orchids. Communications Biology3, 89.

Li H, Liang W, Jia R, Yin C, Zong J, Kong H, Zhang D. 2010. The AGL6-like gene OsMADS6 regulates floral organ and meristem identities in rice. Cell Research20, 299–313.

Mizukami Y, Ma H. 1992. Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell71, 119–131.

Montané M H, Kloppstech K. 2000. The family of light-harvesting-related proteins (LHCs, ELIPs, HLIPs): Was the harvesting of light their primary function? Gene258, 1–8.

Ohmori S, Kimizu M, Sugita M, Miyao A, Hirochika H, Uchida E, Nagato Y, Yoshida H. 2009. MOSAIC FLORAL ORGANS1, an AGL6-like MADS box gene, regulates floral organ identity and meristem fate in rice. The Plant Cell21, 3008–3025.

Pelaz S, Ditta G S, Baumann E, Wisman E, Yanofsky M F. 2000. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature405, 200–203.

Pelaz S, Tapia-López R, Alvarez-Buylla E R, Yanofsky M F. 2001. Conversion of leaves into petals in Arabidopsis. Current Biology11, 182–184.

Peng S M, Luo T, Zhou J Y, Niu B, Lei N F, Tang L, Chen F. 2008. Cloning and quantification of expression levels of two MADS-box genes from Momordica charantiaBiologia Plantarum52, 222–230.

Pinyopich A, Ditta G S, Savidge B, Liljegren S J, Baumann E, Wisman E, Yanofsky M F. 2003. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature424, 85–88.

Purugganan M D. 1997. The MADS-box floral homeotic gene lineages predate the origin of seed plants: Phylogenetic and molecular clock estimates. Journal of Molecular Evolution45, 392–396.

Rijpkema A S, Zethof J, Gerats T, Vandenbussche M. 2009. The petunia AGL6 gene has a SEPALLATA-like function in floral patterning. The Plant Journal60, 1–9.

Rossini S, Casazza A P, Engelmann E C, Havaux M, Jennings R C, Soave C. 2006. Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress. Plant Physiology141, 1264–1273.

Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols3, 1101–1108.

Su Y, Liu J, Liang W, Dou Y, Fu R, Li W, Feng C, Gao C, Zhang D, Kang Z, Li H. 2019. Wheat AGAMOUS LIKE 6 transcription factors function in stamen development by regulating the expression of TaAPETALA3Development146, dev177527.

Suzuki K, Nakanishi H, Bower J, Yoder D W, Osteryoung K W, Miyagishima S Y. 2009. Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thalianaBMC Plant Biology9, 38.

Tao J, Liang W, An G, Zhang D. 2018. OsMADS6 controls flower development by activating rice FACTOR OF DNA METHYLATION LIKE1Plant Physiology177, 713–727.

Theißen G. 2001. Development of floral organ identity: Stories from the MADS house. Current Opinion in Plant Biology4, 75–85.

Theißen G, Melzer R, Rümpler F. 2016. MADS-domain transcription factors and the floral quartet model of flower development: Linking plant development and evolution. Development143, 3259–3271.

Viaene T, Vekemans D, Becker A, Melzer S, Geuten K. 2010. Expression divergence of the AGL6 MADS domain transcription factor lineage after a core eudicot duplication suggests functional diversification. BMC Plant Biology10, 148.

Viitanen P V, Schmidt M, Buchner J, Suzuki T, Vierling E, Dickson R, Lorimer G H, Gatenby A, Soll J. 1995. Functional characterization of the higher plant chloroplast chaperonins. Journal of Biological Chemistry270, 18158–18164.

Wang C, Sun J J, Yang X Y, Wan L, Zhang Z H, Zhang H M. 2023. An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber. Journal of Integrative Agriculture22, 464–470.

Wang T, Liu S, Tian S, Ma T, Wang W. 2022. Light regulates chlorophyll biosynthesis via ELIP1 during the storage of Chinese cabbage. Scientific Reports12, 11098.

Xin T, Tian H, Ma Y, Wang S, Yang L, Li X, Zhang M, Chen C, Wang H, Li H, Xu J, Huang S, Yang X. 2022. Targeted creating new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants. Horticulture Research9, uhab086.

Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. 2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology14, 327.

Yang Y, Fan R, Zhang X J, Li M H, Zhang Y B, Yi H P, Ma M R, Yang Y, Liu B, Liu X W, Ren H Z. 2025. Mutation in CmGhc1 confers the white hypocotyl phenotype in melon (Cucumis melo L.). Journal of Integrative Agriculture24, 4242–4254.

Yoo S K, Hong S M, Lee J S, Ahn J H. 2011. A genetic screen for leaf movement mutants identifies a potential role for AGAMOUS-LIKE 6 (AGL6) in circadian-clock control. Molecules and Cells31, 281–287.

Yu X, Chen G, Guo X, Lu Y, Zhang J, Hu J, Tian S, Hu Z. 2017. Silencing SlAGL6, a tomato AGAMOUS-LIKE6 lineage gene, generates fused sepal and green petal. Plant Cell Reports36, 959–969.

Yu X, Duan X, Zhang R, Fu X, Ye L, Kong H, Xu G, Shan H. 2016. Prevalent exon-intron structural changes in the APETALA1/FRUITFULLSEPALLATAAGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box gene subfamilies provide new insights into their evolution. Frontiers in Plant Science7, 598.

Yu X, Xia S, Xu Q, Cui Y, Gong M, Zeng D, Zhang Q, Shen L, Jiao G, Gao Z, Hu J, Zhang G, Zhu L, Guo L, Ren D, Qian Q. 2020. ABNORMAL FLOWER AND GRAIN 1 encodes OsMADS6 and determines palea identity and affects rice grain yield and quality. Science China Life Sciences63, 228–238.

Zhang X F, Jiang T, Wu Z, Du S Y, Yu Y T, Jiang S C, Lu K, Feng X J, Wang X F, Zhang D P. 2013. Cochaperonin CPN20 negatively regulates abscisic acid signaling in ArabidopsisPlant Molecular Biology83, 205–218.

Zhuang Y, Wei M, Ling C, Liu Y, Amin AK, Li P, Li P, Hu X, Bao H, Huo H, Smalle J, Wang S. 2021. EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in ArabidopsisCell Reports36, 109384.

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