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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (9): 2462-2471.doi: 10.3724/SP.J.1006.2023.24236

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Function analysis of potato StCYP85A3 in promoting germination and root elongation

LIU Jie1,2(), CAI Cheng-Cheng1,2, LIU Shi-Feng1,2, DENG Meng-Sheng3, WANG Xue-Feng2, WEN He1,2, LI Luo-Pin1,2, YAN Feng-Jun1,2, WANG Xi-Yao1,2,*()   

  1. 1State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    2Potato Research and Development Center, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    3College of Biological Engineering, Sichuan University of Light Chemical Industry, Yibin 644000, Sichuan, China
  • Received:2022-10-19 Accepted:2023-02-21 Online:2023-09-12 Published:2023-03-10
  • Supported by:
    Molecular Mechanism and Application of Seed Potato Vigor Regulation(SKL-ZY202203);Sichuan potato Innovation Team Project of National Modern Agricultural Industry Technology System(sccxtd- 2023-09)

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Abstract:

Brassinolide (BR), a kind of phytosterol hormone, plays the significant role in regulating plant growth and development and stress response. The function of StCYP85A3 as the encoding gene of BR synthease in potato remains to be further explored. In this study, the gene was cloned from potato ‘Chuanyu 10’. Bioinformatics analysis showed that this protein had a typical aerobic binding domain for catalyzing BR synthesis and belonged to the CYP85As family. Tissue expression analysis indicated that the relative expression level of this gene was the highest in the bud eye of the tuber and the root, and the expression amount in the bud eye increased with the extension of storage time, and the expression increased rapidly when the dormancy of the tuber bud eye was released. At the same time, the exogenous BR could induce the relative expression level of StCYP85A3 in potato bud eye and root, which significantly promoted the germination of seed potato and the elongation of seedling root. The overexpression of StCYP85A3 in Arabidopsis wild-type and mutant cyp85a2 demonstrated that the seed germination and root elongation of the overexpression strains were earlier than wild type, and the defects of mutant seed germination and plant root growth retardation were remedied by supplementing strains. In addition, exogenous BR did not significantly promote seed germination and root elongation of over expressed strains, but significantly promoted seed germination and root elongation of wild type, mutant, and complementary strains. The above potato and Arabidopsis thaliana experiments showed that StCYP85A3 had the function of promoting germination and root elongation.

Key words: potato, StCYP85A3, BR synthetase, root system, germination

Table 1

Primers used in the study"

基因Gene name 编号Gene ID 引物Primer sequence (5°-3°)
EF1αL Soltu.DM.06G005580 F: CTTGTACACCACGCTAAGGAG
R: GTCAATGCAAACCATTCCTTG
qCYP85A3 Soltu.DM.02G009130.1 F: CCACATACTTGGTTGCCCTAC
R: TGCTCCTCTAATGTGCTTGTG
CYP85A3 Soltu.DM.02G009130.1 F: ATGGCCATTTTCTTGATTGTTTT
R: CTAGTAAGTTGAAACCTTAATC

Fig. 1

Prediction of subcellular location, conservative alignment of protein sequences, and evolutionary tree analysis A: the prediction of subcellular localization; B: protein sequence conservative alignment; C: the analysis of the evolutionary tree; D: gene structure analysis. The stack height is determined by the deviation of the position’s letter emission frequencies from the background frequencies of the letters. As a new feature, the stack width now visualizes both the probability of reaching the state and the expected number of letters the state emits during a pass through the model."

Table 2

Some important cis-acting regulatory elements in the promoters of StCYP85A3"

调控元件
Cis-element		 核心序列
Core sequence		 元件功能描述
Functional description of cis-element		 位置
Position
ABRE CACGTG ABA响应元件
Cis-acting element involved in the abscisic acid responsiveness		 -6, -574
ARE AAACCA 顺式作用调节元件对无氧诱导至关重要
Cis-acting regulatory element essential for the anaerobic induction		 -1467
TGA-element AACGAC 生长素响应元件
Auxin-responsive element		 -205, -438
Box4 ATTAAT 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness		 -12, -112, -818, -1082, -1442
GT1-motif GGTTAA 光响应元件
Cis-acting element for light responsive		 -91
G-box TACGTG 光响应元件
Cis-acting element for light responsive		 -6, -574
CAT-box GCCACT 与分生组织表达相关的顺式作用调节元件
Cis-acting regulatory element related to meristem expression		 -245

Fig. 2

Analysis of CYP85A3 tissue expression and expression of CYP85A3 in the eye bud of C10 during storage A: CYP85A3 tissue expression; B: CYP85A3 expression during C10 storage. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters represent significant differences between tissues and after storage at P < 0.05."

Fig. 3

Effect of BR exogenous application on potato tuber germination and root length A: the effects of different concentrations of BR on potato tuber germination rate; B: the effect of different concentrations of BR; C: the effect of BR treatment on the expression of StCYP85A3 in potato bud eyes; D: the effect of BR treatment on the expression of StCYP85A3 in potato seedling roots. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters represent significant differences at P < 0.05."

Fig. 4

Overexpression, mutant, and supplementary germination rate A: the statistics of germination rate of different strains of Arabidopsis thaliana on MS medium; B: the statistics of germination rate of different lines of Arabidopsis thaliana on MS medium containing 50 nmol L-1 BR; C: the germination of Arabidopsis thaliana of different strains on MS medium after 3 days of planting; D: germination of Arabidopsis thaliana of different strains on MS medium containing 50 nmol L-1 BR after 60 hours of planting. The error line represents the standard deviation. The 60 hours data in Fig. A and Fig. B are jointly analyzed for significance. Student’s t-test is used for statistical analysis. Different lowercase letters indicate significant differences in germination of different strains at P < 0.05."

Fig. 5

Overexpression, mutant, and root length A: the statistics of root length of different lines of Arabidopsis thaliana on MS and MS medium containing 50 nmol L-1 BR; B: root length phenotypes of different Arabidopsis lines on MS medium; C: changes of StCYP85A3 expression in Arabidopsis thaliana roots treated with 50 nmol L-1 BR over time; D: root length phenotype of Arabidopsis thaliana lines treated with 50 nmol L-1 BR after 10 days of germination. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters indicate significant differences in root length and the relative expression level at P < 0.05."

[1] 荐红举, 张梅花, 尚丽娜, 王季春, 胡柏耿, Vadim K, 吕典秋. 利用WGCNA筛选马铃薯块茎发育候选基因. 作物学报, 2022, 48: 1658-1668.
doi: 10.3724/SP.J.1006.2022.14115
Jian H J, Zhang M H, Shang L N, Wang J C, Hu B G, Vadim K, Lyu D Q. Screening candidate genes involved in potato tuber development using WGCNA. Acta Agron Sin, 2022, 48: 1658-1668. (in Chinese with English abstract)
[2] 何蒲明, 狄书非. 生态安全与粮食安全并重导向下马铃薯主粮化发展路径研究. 农业经济, 2019, (6): 3.
He P M, Di S F. Research on the development path of potato as staple food with equal emphasis on ecological security and food security. Agric Econ, 2019, (6): 3. (in Chinese)
[3] Li L, Deng M S, Lyu C, Zhang J, Peng J, Cai C C, Yang S M, Lu L M, Ni S, Liu F. Quantitative phosphoproteomics analysis reveals that protein modification and sugar metabolism contribute to sprouting in potato after BR treatment. Food Chem, 2020, 325: 126-139.
[4] Wang L, Liu J, Shen Y T, Pu R L, Hou M Y, Wei Q, Zhang X Z, Li G S, Ren H Y, Wu G. Brassinosteroids synthesised by CYP85A/A1 but not CYP85A2 function via a BRI1-like receptor but not via BRI1 in Picea abies. J Exp Bot, 2020, 5: 5.
[5] Katsumata T, Hasegawa A, Fujiwara T, Komatsu T, Kawaide H. Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone. Brassinosteroids. Biosci Biotechnol Biochem, 2008, 72: 210-217.
[6] Pereze V H, Nidia S L, Vielle C J P. CYP85A1 is required for the initiation of female gametogenesis in Arabidopsis thaliana. Plant Signal Behav, 2011, 6: 321-326.
doi: 10.4161/psb.6.3.13206
[7] Jin Y L, Tang R J, Wang H H, Jiang C M, Bao Y, Yang Y, Liang M X, Sun Z C, Kong F J, Li B, Zhang H X. Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees. Plant Biotechnol J, 2017, 15: 29-32.
[8] Nomura T, Kushiro T, Yokota T, Kamiya Y, Bishop G J, Yamaguchi S. The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis. J Biol Chem, 2005, 280: 73-79.
doi: 10.1074/jbc.M409795200
[9] 邹雪, 丁凡, 余金龙, 彭洁, 邓孟胜, 王宇, 刘丽芳, 余韩开宗, 陈年伟, 王西瑶. 挥发性抑芽物质对马铃薯块茎萌芽的影响及其作用机制. 作物学报, 2019, 45: 235-247.
doi: 10.3724/SP.J.1006.2019.84063
Zou X, Ding F, Yu J L, Peng J, Deng M S, Wang Y, Liu L F, Yu-Han K Z, Chen N W, Wang X Y. Suppression mechanism of volatile sprout-inhibitors on potato tuber sprouting. Acta Agron Sin, 2019, 45: 235-247. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2019.84063
[10] 邹雪, 邓孟胜, 李立芹, 余金龙, 丁凡, 黄雪丽, 彭洁, 帅禹, 蔡诚诚, 王西瑶. 油菜素内酯合成和信号转导基因在马铃薯块茎贮藏期间的表达变化及对萌芽的影响. 作物学报, 2017, 43: 811-820.
Zou X, Deng M S, Li L Q, Yu J L, Ding F, Huang X L, Peng J, Shuai Y, Cai C C, Wang X Y. Expression changes of genes related to brassinosteroid biosynthesis and signal transduction during potato storage and its effect on tuber sprouting. Acta Agron Sin, 2017, 43: 811-820. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2017.00811
[11] 马琛. 基于高通量测序半夏变异珠芽转录组研究. 浙江理工大学硕士学位论文, 浙江杭州, 2018.
Ma C. Based on High-throughput Sequencing of Pinellia ternate Variant Bulbil Transcriptome. MS Thesis of Zhejiang Sci-Tech University, Hangzhou, Zhejiang, China, 2018. (in Chinese with English abstract)
[12] Chen J, Liu S S, Kohler A, Yan B, Luo H M, Chen M X, Guo S X. iTRAQ and RNA-Seq analyses provide new insights into regulation mechanism of symbiotic germination of dendrobium officinale seeds (Orchidaceae). J Proteome Res, 2017, 16: 174-187.
[13] 张松, 黄波, 夏学峰, 孙之荣. 蛋白质亚细胞定位的生物信息学研究. 生物化学与生物物理进展, 2007, 34: 573-579.
Zhang S, Huang B, Xia X F, Sun Z R. Bioinformatics research in subcellular localization of proteins. Prog Biochem Biophys, 2007, 34: 573-579. (in Chinese with English abstract)
[14] 范敏, 金黎平, 黄三文, 谢开云, 刘庆昌, 屈冬玉. 马铃薯SoFtsH基因全长cDNA克隆与在干旱条件下表达研究. 作物学报, 2007, 33: 1748-1754.
Fan M, Jin L P, Huang S W, Xie K Y, Liu C Q, Qu D Y. Cloning and expression of SoFtsH gene in potato under drought. Acta Agron Sin, 2007, 33: 1748-1754 (in Chinese with English abstract).
[15] 李素云, 李佳, 王莉莉, 钟嘉宏, 陈青, 熊文芳, 曾小微, 贺修胜. STGC3基因启动子生物信息学分析及载体构建. 中南医学科学杂志, 2017, 45(1): 48-53.
Li S Y, Li J, Wang L L, Zhong J H, Chen Q, Xiong W F, Zeng X W, He X S. Bioinformatics analysis and vector construction of STGC3 gene promoter. Med Sci J Central South China, 2017, 45(1): 48-53. (in Chinese with English abstract)
[16] 王怡, 于月华, 杨成元, 陈全家, 倪志勇. GbTCP10基因植物表达载体的构建及拟南芥转化. 分子植物育种, 2020, 18: 144-149.
Wang Y, Yu Y H, Yang C Y, Chen Q J, Ni Z Y. Construction of plant expression vector of GbTCP10 gene and transformation of Arabidopsis, Mol Plant Breed, 2020, 18: 144-149. (in Chinese with English abstract)
[17] 孟颖, 邢蕾蕾, 曹晓红, 郭光艳, 柴建芳, 秘彩莉. 小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能. 作物学报, 2022, 48: 63-75.
doi: 10.3724/SP.J.1006.2022.01100
Meng Y, Xing L L, Cao X H, Guo G Y, Chai J F, Bei C L. Cloning of wheat Ta4CL1 gene and its function in promoting the growth and lignin deposition of transgenic Arabidopsis thaliana. Acta Agron Sin, 2022, 48: 63-75. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2022.01100
[18] 李胜婷, 徐远芳, 常玮, 刘亚俊, 谷嫄, 朱红, 李加纳, 卢坤. Bna. C02SWEET15通过光周期途径正向调控油菜开花时间. 作物学报, 2022, 48: 1938-1947.
doi: 10.3724/SP.J.1006.2022.14155
Li S T, Xu Y F, Chang W, Liu Y J, Gu Y, Zhu H, Li J N, Lu K. Bna.C02SWEET15 positively regulates the flowering time of rapeseed through photoperiodic pathway. Acta Agron Sin, 2022, 48: 1938-1947. (in Chinese with English abstract)
[19] 邸雪妮, 邓孟胜, 邹雪, 李雅楠, 倪苏, 王西瑶. BR相关基因在低温调控马铃薯萌芽中的响应分析. 分子植物育种, 2018, 16: 4143-4150.
Di X N, Deng M S, Zou X, Li Y A, Ni S, Wang X Y. Response analysis of BR related genes in low temperature regulation of potato germination. Mol Plant Breed, 2018, 16: 4143-4150. (in Chinese with English abstract)
[20] Cai Z Y, Liu J J, Wang H J, Yang C J, Chen Y X, Li Y C, Pan S J, Dong R, Tang G L, Barajas L J D, Fujii H, Wang X L. GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis. Proc Natl Acad Sci USA, 2014, 11: 11-16.
doi: 10.1073/pnas.11.1.11
[21] Wang L, Liu J, Shen Y T, Pu R L, Hou M Y, Wei Q, Zhang X Z, Li G S, Ren H Y, Wu G. Brassinosteroids synthesised by CYP85A/A1 but not CYP85A2 function via a BRI1-like receptor but not via BRI1 in Picea abies. J Exp Bot, 2021, 72: 1748-1763.
doi: 10.1093/jxb/eraa557 pmid: 33247718
[22] 唐晓, 邓孟胜, 邹雪, 祝渊智, 王西瑶. 马铃薯StDWF1基因克隆及表达分析. 浙江农业学报, 2018, 30: 909-917.
doi: 10.3969/j.issn.1004-1524.2018.06.04
Tang X, Deng M S, Zou X, Zhu Y Z, Wang X Y. Cloning and expression analysis of StDWF1 gene in potato. Acta Agric Zhejiangensis, 2018, 30: 909-917. (in Chinese with English abstract)
[23] Zhao W Y, Bai Q Z, Zhao B L, Wu Q, Wang C Q, Liu Y, Yang T Q, Liu Y, He H, Du S S. The geometry of the compound leaf plays a significant role in the leaf movement of Medicago truncatula modulated by mtdwarf4a. New Phytol, 2021, 230: 475-484.
doi: 10.1111/nph.v230.2
[24] Wang L, Wang Z, Xu Y Y, Joo S H, Kim S K, Xue Z, Xu Z H, Wang Z Y, Chong K. OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice. Plant J, 2010, 57: 498-510.
doi: 10.1111/tpj.2009.57.issue-3
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