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| Functional Identification and Analysis of Ph4CL13 Gene in Petunia(Petunia hybrida) |
| MIAO Yun-Feng*, HU Xu-Hao*, LIN Bin-Ru, ZHONG Shi-Wei** |
| School of Landscape Architecture, Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang A & F University, Hangzhou 311300, China |
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Abstract The main pigment responsible for the coloration of plant petals is anthocyanin. Based on previous transcriptome data from petunias, the highly expressed 4-coumarate CoA ligase 13 (Ph4CL13) gene was identified during the bud coloring stage, suggesting its involvement in anthocyanin synthesis. Anthocyanin primarily determines petal color in plants. To investigate the role of Ph4CL13 in anthocyanin synthesis, this study used Petunia hybrida 'Ultra' as the material. The 1 653 bp cDNA fragment of Ph4CL13 (Peaxi162Scf00089g00045.1) was isolated from the petunia genome. Phylogenetic analysis revealed a close relationship between Ph4CL13 and Fa4CL-1 and Fa4CL-2. qPCR analysis revealed that Ph4CL13 was highly expressed during the coloring stages, suggested its involvement in anthocyanin synthesis and impact on petunia flower coloration. Silencing Ph4CL13 via virus-induced gene silencing (VIGS), the results showed that the corollas of petunia turned white, and the level of anthocyanin significantly decreased in Ph4CL13-silenced plant compared to control. qPCR analyses demonstrated that Ph4CL13 silencing transcriptionally repressed several anthocyanin biosynthesis genes, including chalconesynthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid synthase (FLS), suggested that Ph4CL13 played a critical role in the anthocyanin synthesis stage of flower coloring process, that might be a potential complex feedback mechanism among these genes related to anthocyanin synthesis. This study preliminarily revealed the important role of Ph4CL13 in anthocyanin synthesis, providing a reference for further exploring its molecular mechanisms in flower color development.
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Received: 16 June 2023
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Corresponding Authors:
** zsw1105@zafu.edu.cn
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| About author:: * These authors contributed equally to this work |
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[1] 洪燕红, 叶清华, 李泽坤, 等. 2021. 红花草莓'莓红'花瓣花色苷积累及其MYB基因的表达分析[J]. 园艺学报, 48(08): 1470-1484.
(Hong Y H, Ye Q H, Li Z K, et al.2021. Accumulation of anthocyanins in red-flowered strawberry 'Meihong' petals and expression analysis of MYB gene[J]. Acta Horticulturae Sinica, 48(08): 1470-1484.)
[2] 李逸, 徐欢欢, 张宇辰, 等. 2023. 大葱4-香豆酸辅酶A连接酶基因Af4CL的克隆及表达分析[J]. 农业生物技术学报, 30(02): 273-281.
(Li Y, Xu H H, Zhang Y C, et al.2023. Cloning and expression analysis of 4-coumaric acid coenzyme A ligase gene Af4CL in Allium fistulosum[J]. Journal of Agricultural Biotechnology, 30(02): 273-281.)
[3] 刘和平. 2016. 矮牵牛PhACC1、PhACC2和PhAAE13在花青素合成中的作用研究[D]. 硕士学位论文, 华南农业大学, 导师: 陈国菊, pp. 40-43.
(Liu H P.Functional analysis of PhACC1, PhACC2 and PhAAE13 during the anthocyanin biosynthesis in the petunia[D]. Thesis for M.S., South China Agricultural University, Supervisor: Chen G J, pp. 40-43)
[4] 申晚霞, 王志彬, 薛杨, 等. 2019. 柑橘4CL基因家族的结构及其功能分析[J]. 园艺学报, 46(06): 1068-1078.
(Shen W X, Wang Z B, Xue Y, et al.2019. Characterization of 4-coumarate: CoA Ligase (4CL) gene family in Citrus[J]. Acta Horticulturae Sinica, 46(06): 1068-1078.)
[5] 王会平, 遇玲, 邹世慧, 等. 2012. 利用amiRNA技术沉默矮牵牛查尔酮合成酶基因[J]. 园艺学报, 39(12): 2491-2498.
(Wang H P, Yu L, Zou S H, et al.2012. Silencing of chalcone synthase genes by artificial microRNA in Petunia[J]. Acta Horticulturae Sinica, 39(12): 2491-2498.)
[6] 谢海娟, 范希德, 叶广继, 等. 2019. 马铃薯St4CL的克隆及表达分析[J]. 生物技术通报, 35(11): 1-8.
(Xie H J, Fan X D, Ye G J, et al.2019. Cloning and expression analysis of St4CL gene in Solanum tuberosum[J]. Biotechnology Bulletin, 35(11): 1-8.)
[7] 张蕾, 林晓, 罗赟, 等. 2015. RNAi沉默Fa4CL基因对草莓果实花色苷代谢的影响[J]. 果树学报, 32(03): 434-439.
(Zhang L, Lin X, Luo Y, et al.2015. Influences of RNAi-induced Fa4CL silencing on anthocyanin metabolism in strawberry fruit[J]. Journal of Fruit Science, 32(03): 434-439.)
[8] 钟秀来, 赵倩, 朱顺华, 等. 2023. 芹菜Ag4CL1基因克隆及功能分析[J]. 分子植物育种, 1-13.
(Zhong X L, Zhao Qi, Zhu S H, et al.Cloning and expression analysis of Ag4CL1 gene in Apium graveolens[J]. Molecular Plant Breeding, 1-13.)
[9] 祝钦泷. 2004. 转查尔酮异构酶(CHI)基因矮牵牛花色改变及其花器官变异的研究[D]. 硕士学位论文, 西南农业大学, 导师: 李名扬, pp. 52-53.
(Zhu Q L.2004. Expression of chalcone isomerase gene caused changes of flower color and variations of flower organ in transgenic petunia[D]. Thesis for M.S., Southwest Agricultural University, Supervisor: Li M Y, pp. 52-53.)
[10] Chen G J, Liu H P, Wei Q, et al.2016. The acyl-activating enzyme PhAAE13 is an alternative enzymatic source of precursors for anthocyanin biosynthesis in petunia flowers[J]. Journal of Experimental Botany, 68(3): 457-467.
[11] Chen X H, Su W L, Zhang H, et al.2020. Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content[J]. Plant Physiology and Biochemistry, 155: 697-708.
[12] Chen Z Y, Yuan J W, Yao Y, et al.2023. PhAAT1, encoding an anthocyanin acyltransferase, is transcriptionally regulated by PhAN2 in petunia[J]. Physiologia Plantarum, 175(1): e13851.
[13] Ehlting J, Büttner D, Wang Q, et al.1999. Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms[J]. The Plant Journa, 19(1): 9-20.
[14] Gerats T, Vandenbussche M.2005. A model system for comparative research: Petunia[J]. Trends in Plant Science, 10(5): 251-256.
[15] He F, Mu L, Yan G L, et al.2010. Biosynthesis of anthocyanins and their regulation in colored grapes[J]. Molecules, 15(12): 9057-9091.
[16] Hichri I, Barrieu F, Bogs J, et al.2011. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway[J]. Journal of Experimental Botany, 62(8): 2465-2483.
[17] Holton T A, Cornish E C.1995. Genetics and biochemistry of anthocyanin biosynthesis[J]. The Plant Cell, 7(7): 1071-1083.
[18] Huang C Y, Wen W E, Li Q Q, et al.2023. Identification, characterization and expression analysis of the 4-coumarate-coA ligase gene family in Bletilla striata[J]. Gene Reports, 32: 101785.
[19] Kumar A, Ellis B E.2003. 4-Coumarate: CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression[J]. Plant Molecular Biology, 51(3): 327-340.
[20] Ma Z H, Nan X T, Li W F, et al.2023. Comprehensive genomic identification and expression analysis 4CL gene family in apple[J]. Gene, 858: 147197.
[21] Mallona I, Lischewski S, Weiss J, et al.2010. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J]. BMC Plant Biology, 10(1): 4.
[22] Meng J, Li C N, Zhao M L, et al.2018. Lignin biosynthesis regulated by the antisense 4CL gene in alfalfa[J]. Czech Journal of Genetics and Plant Breeding, 54(1): 26-29.
[23] Nakajima T, Matsubara K, Kodama H, et al.2005. Insertion and excision of a transposable element governs the red floral phenotype in commercial petunias[J]. Theoretical and Applied Genetics, 110: 1038-1043.
[24] Reinold S, Hauffe K D, Douglas C J.1993. Tobacco and parsley 4-Coumarate: Coenzyme A ligase genes are temporally and spatially regulated in a cell type-specific manner during tobacco flower development[J]. Plant Physiology, 101(2): 373-383.
[25] Soltani B M, Ehlting J, Hamberger B, et al.2006. Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members[J]. Planta, 224(5): 1226-1238.
[26] Spitzer B, Zvi M M B, Ovadis M, et al.2007. Reverse genetics of floral scent: Application of tobacco rattle virus-based gene silencing in petunia[J]. Plant Physiology, 145(4): 1241-1250.
[27] Tanaka Y, Fukui Y, Fukuchi-Mizutani M, et al.1995. Molecular cloning and characterization of Rosa hybrida dihydroflavonol 4-reductase gene[J]. Plant and Cell Physiology, 36(6): 1023-1031.
[28] Tang N, Cao Z Y, Yang C, et al.2021. A R2R3-MYB transcriptional activator LmMYB15 regulates chlorogenic acid biosynthesis and phenylpropanoid metabolism in Lonicera macranthoides[J]. Plant Science, 308: 110924.
[29] Wang C H, Yu J, Cai Y X, et al.2016. Characterization and functional analysis of 4-Coumarate: CoA Ligase genes in mulberry[J]. PLOS ONE, 11: e0155814.
[30] Wang Y Y, Guo L H, Zhao Y J, et al.2022. Systematic analysis and expression profiles of the 4-Coumarate: CoA ligase (4CL) gene family in pomegranate (Punica granatum L.)[J]. International Journal of Molecular Sciences, 23(7): 3509.
[31] Wei H, Chen X, Zong X, et al.2015. Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the red and yellow fruits of sweet cherry (Prunus avium L.)[J]. PLOS ONE, 10(3): e0121164.
[32] Wei H, Xu C, Movahedi A, et al.2019. Characterization, expression profiling, and functional analyses of a 4CL-Like gene of Populus trichocarpa[J]. Processes, 7(1): 45.
[33] Yuan J W, Zhong S W, Long Y, et al.2022. Shikimate kinase plays important roles in anthocyanin synthesis in petunia[J]. International Journal of Molecular Sciences, 23(24): 15964.
[34] Zhao H, Chen G, Sang L, et al.2021. Mitochondrial citrate synthase plays important roles in anthocyanin synthesis in petunia[J]. Plant Science, 305: 110835.
[35] Zhong J, Qing J, Wang Q, et al.2022. Genome-wide identification and expression analyses of the 4-Coumarate: CoA ligase (4CL) gene family in Eucommia ulmoides[J]. Forests, 13(8): 1253. |
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