Cloning and Functional Analysis of LaCKX1 Gene Related to Regulation of Floret Number in Lavandula angustifolia

doi:10.13560/j.cnki.biotech.bull.1985.2026-0310

Abstract

Abstract:

Objective Cytokinin oxidase/dehydrogenase (CKX) irreversibly degrades cytokinin (CK) and plays a key regulatory role in plant flower bud differentiation. This study explored the function of the LaCKX1 gene during Lavandula angustifolia spike development, providing a theoretical basis for revealing the regulatory mechanism of the cytokinin signaling pathway on floret number formation in L. angustifolia. Method The LaCKX1 gene was cloned from the flower spike cDNA of L. angustifolia cultivar ‘Xinnongxun No.1’. Its protein physicochemical properties and phylogenetic relationships were analyzed using bioinformatics methods. Subcellular localization of the LaCKX1 protein was determined by transient expression in Nicotiana benthamiana. The expression levels of LaCKX1 in different L. angustifolia varieties, tissues, and spike developmental stages were examined by real-time quantitative PCR (RT-qPCR). The function of LaCKX1 and its effect on endogenous CK content were verified through genetic transformation in Arabidopsis thaliana and L. angustifolia. Result The LaCKX1 gene (coding sequence of 1 575 bp, encoding 524 amino acids) was successfully cloned, and its encoded protein was localized to the cell membrane. LaCKX1 exhibited a distinct tissue-specific expression pattern, with high transcript levels in flower spikes, particularly during the early stages of flower spike development. Its expression was significantly higher in the low-grain cultivar than in the high-grain cultivar at the S1 stage. Furthermore, LaCKX1 overexpression in Arabidopsis resulted in decreased endogenous CK content, leading to phenotypic changes including longer roots, reduced plant height, and fewer flowers. Similarly, LaCKX1 overexpression reduced CK content in L. angustifolia leaves, whereas gene silencing increased CK content in newly formed leaves. Conclusion LaCKX1 may negatively regulate CK biosynthesis in L. angustifolia, thereby participating in the regulation of its flower spike development.

Key words: Lavandula angustifolia, LaCKX1 gene, cytokinin, cytokinin oxidase/dehydrogenase, floret number, gene cloning, functional verification

YUE Li-ping, WANG Ai-fan, WANG Shan, LI Xue-long, CHEN Yu-xiang, SU Xiu-juan. Cloning and Functional Analysis of LaCKX1 Gene Related to Regulation of Floret Number in Lavandula angustifolia[J]. Biotechnology Bulletin, 2026, 42(9): 238-247.

Figures/Tables 7

Fig. 1 Different flowering stages of L. angustifoliaS1: Early stage of panicle development. S2: First stem node begins to develop. S3: First stem node is half-developed. S4: First stem node is fully developed. The same below

Fig. 2 Multiple sequence alignment (A) and phylogenetic tree (B) of the LaCKX1 protein with CKX proteins from other speciesThe black, red, and blue parts indicate homology = 100%, ≥75%, and ≥50%, respectively. Green boxes are the FAD-binding domain, and red boxes are the cytokinin-binding domain

Fig. 3 Subcellular localization of LaCKX1

Fig. 4 Expression levels of LaCKX1 in different tissues and at different flower spike developmental stagesDifferent letters indicate significant differences at the 0.05 level (P<0.05). The same below

Fig. 5 PCR identification of positive transgenic LaCKX1Arabidopsis, comparison of phenotypic differences, and changes in CK contentA: PCR identification of positive transgenic Arabidopsis thaliana (M: 2000 DNA marker. 1-3: Resistant seedlings of LaCKX1-overexpressing Arabidopsis. 4: Positive control. 5: Negative control). B: Comparison of root length between LaCKX1-overexpressing Arabidopsis lines and wild-type (WT) seedlings at 14 d of culture. C: Plant height phenotype at 35 d of culture. D: Statistical analysis of plant height at 35 d of culture. E: Determination of plant cytokinin (CK) content

Fig. 6 Detection of gene expression and CK content in LaCKX1-overexpressed (A, B) and gene-silenced (C, D) L. angustifoliaA: Expression levels of LaCKX1 in LaCKX1-overexpressing L. angustifolia lines and wild-type (WT). B: Cytokinin (CK) contents in LaCKX1-overexpressing L. angustifolia lines and wild-type (WT). C: Expression level of the target gene in L. angustifolia gene-silenced lines. D: CK content in L. angustifolia gene-silenced lines

Fig. 7 Model diagram of LaCKX1 regulating floret grain number in L. angustifolia: Promotional effect. : Inhibitory effect. : CK

References 30

[1]	Al-Ansari MM, Andeejani AMI, Alnahmi E, et al. Insecticidal, antimicrobial and antioxidant activities of essential oil from Lavandula latifolia L. and its deterrent effects on Euphoria leucographa [J]. Ind Crops Prod, 2021, 170: 113740.
[2]	Kitagawa M, Jackson D. Control of meristem size [J]. Annu Rev Plant Biol, 2019, 70: 269-291.
[3]	Lu JQ, Jiang ZJ, Chen JY, et al. SET DOMAIN GROUP 711-mediated H3K27 me3 methylation of cytokinin metabolism genes regulates organ size in rice [J]. Plant Physiol, 2024, 194(4): 2069-2085.
[4]	余义和, 李秀珍, 陈迪新, 等. ‘巨峰’葡萄细胞分裂素氧化酶基因CKX3的克隆与表达分析 [J]. 园艺学报, 2015, 42(7): 1233-1240.
	Yu YH, Li XZ, Chen DX, et al. Isolation and expression analysis of cytokinin oxidase gene CKX3 in kyoho grapevine [J]. Acta Hortic Sin, 2015, 42(7): 1233-1240.
[5]	边林, 张岩, 霍晓伟, 等. 紫花苜蓿CKX基因家族鉴定及其对非生物胁迫的响应 [J]. 草业学报, 2025, 34(11): 125-135.
	Bian L, Zhang Y, Huo XW, et al. Identification of the CKX gene family in alfalfa and its responses to abiotic stress [J]. Acta Prataculturae Sin, 2025, 34(11): 125-135.
[6]	Ashikari M, Sakakibara H, Lin SY, et al. Cytokinin oxidase regulates rice grain production [J]. Science, 2005, 309(5735): 741-745.
[7]	Li J, Nie X, Tan JLH, et al. Integration of epigenetic and genetic controls of seed size by cytokinin in Arabidopsis [J]. Proc Natl Acad Sci U S A, 2013, 110(38): 15479-15484.
[8]	Bartrina I, Otto E, Strnad M, et al. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana [J]. Plant Cell, 2011, 23(1): 69-80.
[9]	Li SY, Zhao BR, Yuan DY, et al. Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression [J]. Proc Natl Acad Sci U S A, 2013, 110(8): 3167-3172.
[10]	Zhang W, Peng KX, Cui FB, et al. Cytokinin oxidase/dehydrogenase OsCKX11 coordinates source and sink relationship in rice by simultaneous regulation of leaf senescence and grain number [J]. Plant Biotechnol J, 2021, 19(2): 335-350.
[11]	Werner T, Motyka V, Laucou V, et al. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity [J]. Plant Cell, 2003, 15(11): 2532-2550.
[12]	Han YY, Yang HB, Jiao YL. Regulation of inflorescence architecture by cytokinins [J]. Front Plant Sci, 2014, 5: 669.
[13]	Jing WK, Gong FF, Liu GQ, et al. Petal size is controlled by the MYB73/TPL/HDA19-miR159-CKX6 module regulating cytokinin catabolism in Rosa hybrida [J]. Nat Commun, 2023, 14: 7106.
[14]	任世恒. 繁殖方式、收获时期及方式对薰衣草农艺性状、精油产量及品质的影响 [D]. 乌鲁木齐: 新疆农业大学, 2024.
	Ren SH. Effects of reproduction modes, harvesting period and modes on agronomic traits, yield and quality of essential oil in lavender [D]. Urumqi: Xinjiang Agricultural University, 2024.
[15]	王振昱. 薰衣草萜类花香合成关键酶DXS、DXR基因的功能验证 [D]. 乌鲁木齐: 新疆农业大学, 2023.
	Wang ZY. Functional verification of the key enzymes DXS and DXR genes in the synthesis of terpenoid floral aroma from lavender [D]. Urumqi: Xinjiang Agricultural University, 2023.
[16]	李鹏飞, 王爱凡, 黄佳慧, 等. 薰衣草花青素合成相关基因LaCHS的克隆与功能分析 [J]. 浙江农林大学学报, 2025, 42(5): 1048-1058.
	Li PF, Wang AF, Huang JH, et al. Cloning and functional analysis of LaCHS gene related to anthocyanin biosynthesis in Lavandula angustifolia [J]. J Zhejiang A F Univ, 2025, 42(5): 1048-1058.
[17]	D'Ario M, Sablowski R. Cell size control in plants [J]. Annu Rev Genet, 2019, 53: 45-65.
[18]	Powell AE, Lenhard M. Control of organ size in plants [J]. Curr Biol, 2012, 22(9): R360-R367.
[19]	Chen SF, Lou SL, Zhao XC, et al. Ectopic expression of a male fertility gene, LOGL8 represses LOG and hinders panicle and ovule development [J]. Crop J, 2022, 10(6): 1665-1673.
[20]	Chun Y, Fang JJ, Savelieva EM, et al. The cytokinin receptor ohk4/OsHK4 regulates inflorescence architecture in rice via an ideal plant architecture1/wealthy farmer’s panicle-mediated positive feedback circuit [J]. Plant Cell, 2023, 36(1): 40-64.
[21]	Werner T, Schmülling T. Cytokinin action in plant development [J]. Curr Opin Plant Biol, 2009, 12(5): 527-538.
[22]	Sharma A, Prakash S, Chattopadhyay D. Killing two birds with a single stone—genetic manipulation of cytokinin oxidase/dehydrogenase (CKX) genes for enhancing crop productivity and amelioration of drought stress response [J]. Front Genet, 2022, 13: 941595.
[23]	Wang HM, Tong XH, Tang LQ, et al. Rlb (rice lateral branch) recruits prc2-mediated h3k27 tri-methylation on OsCKX4 to regulate lateral branching [J]. Plant Physiol, 2022, 188(1): 460-476.
[24]	Hwang I, Sheen J, Müller B. Cytokinin signaling networks [J]. Annu Rev Plant Biol, 2012, 63: 353-380.
[25]	Skalák J, Vercruyssen L, Claeys H, et al. Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis [J]. Plant J, 2019, 97(5): 805-824.
[26]	Li YX, Zhu GS, Sun HN, et al. Genome-wide analysis of LOG family genes in castor and RcLOG5 enhances drought, salt, and cold stress tolerance in Arabidopsis thaliana [J]. Gene, 2024, 913: 148398.
[27]	Zürcher E, Müller B. Cytokinin synthesis, signaling, and function—advances and new insights [M]//International Review of Cell and Molecular Biology. Amsterdam: Elsevier, 2016: 1-38.
[28]	Nisler J, Pěkná Z, Končitíková R, et al. Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency [J]. J Exp Bot, 2022, 73(14): 4806-4817.
[29]	禹一箭, 李振宇, 张金青. 植物细胞分裂素氧化/脱氢酶基因家族研究进展 [J]. 植物生理学报, 2026, 62(2): 125-138.
	Yu YJ, Li ZY, Zhang JQ. Research progress on the cytokinin oxidase/dehydrogenase gene family in plants [J]. Plant Physiol J, 2026, 62(2): 125-138.
[30]	Ding WN, Tong HS, Zheng WJ, et al. Isolation, characterization and transcriptome analysis of a cytokinin receptor mutant Osckt1 in rice [J]. Front Plant Sci, 2017, 8: 88.