1 College of Horticulture Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao 066600, China; 2 College of Horticulture/Institute of Genetics & Breeding in Horticultural Plants, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
Abstract：The number of scapes is an important index to measure the quality and price of narcissus (Narcissus tazetta) bulbs. Because it is hard to strict control ethylene concentration, the traditional method for narcissus bulbs fumigation may cause bad effect on flower bud induction. In this study, transcriptome with deep sequencing was used on the ethylene (ETH)/1-methylcyclopropene (1-MCP) treated narcissus to reveal the molecular mechanism of the floral initiation of narcissus. A scientific method of narcissus bulbs treatment have been obtained in our study. Narcissus bulbs were incubated with 200 μL/L of ethylene for 5 h in a sealed container and these bulbs were treated again two days later. Ethylene treatment promotes the floral bud differentiation of narcissus and doubles the number of scapes and flowers. The results of physiological and biochemical indexes showed that exogenous ethylene application promoted DNA replication, gene expression, protein synthesis, sugar metabolism, plant hormone signal transduction, increased the level of protein, peroxidase (POD) activity, indole-3-acetic acid (IAA), and zeatin (ZA) level. De novo transcriptome and RNA-Seq analysis of three treatment groups showed that 65 898 unigenes were obtained in all samples, including 55 793(control group), 57 321(1-MCP), and 64 350(ETH), with average length is 692 bp. Gene Ontology (GO) analysis showed that exogenous ethylene promotes the expression of most genes in all terms. Through differential expression analysis, 62 differentially expressed genes (DEGs) related to flowering were screened out: four genes in starch and sucrose metabolism, nine genes in polyamine synthesis and transport, eleven genes in lignin synthesis and transport, thirty-one flowering-related genes, and seven other regulation genes. Key genes of COL14, FRL3, VRN1, FPA in the photoperiod pathway, vernalization pathway, autonomous pathway had significant difference in expression compared to the control group. Twelve genes correlated with flowering were selected and confirmed by qRT-PCR analysis and the expression profiles were consistent with the RNA-Seq results. These DEGs might have vital function on the floral bud differentiation of narcissus. In this study, a scientific technique of narcissus bulbs treated with ETH has been conducted, and the physiological, biochemical and molecular mechanism of ethylene had been discussed. The study will be helpful for better understanding the floral bud differentiation of narcissus, as well as guides the narcissus production.
[1] 陈炫, 陶忠良, 吴志祥, 等. 2007. 多效唑、乙烯利对妃子笑荔枝成花的影响[J]. 热带农业科学, 27(5): 8-10. (Chen X, Tao Z L, Wu Z X, et al.2007. Effects of PP333 and ethrel on blossom of litchi 'Feizixiao'[J]. Chinese Journal of Tropical Agriculture, 27(5): 8-10.) [2] 陈炫, 陶忠良, 吴志祥, 等. 2012. 多效唑+乙烯利对妃子笑荔枝内源激素及碳氮营养的影响[J]. 江西农业大学学报, 34(1): 27-33. (Chen X, Tao Z L, Wu Z X, et al.2012. Effect of PP333 and ethrel treatment on endogenous hormones and carbon and nitrogen nutrients in Feizixiao litchi[J]. Acta Agriculturae Universitatis Jiangxiensis, 34(1): 27-33.) [3] 方舟. 2017. 水仙‘金玉’花芽分化研究[D]. 硕士学位论文, 福建农林大学, 导师: 陈晓静. pp. 24-26. (Fang Z.2017. Morphological study on flower bud differentiation of Narcissus tazetta L. var. chinensis[D]. Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Chen X J, pp. 24-26.) [4] 冯莹. 2014. 中国水仙(Narcissus tazetta var. chinensis)芽休眠的分子机制研究[D]. 博士学位论文, 福建农林大学, 导师: 潘东明. pp. 87-88. (Feng Y.2014. The molecular mechanism of bud dormancy in Narcissus tazetta var. chinensis[D]. Thesis for Ph.D., Fujian Agriculture and Forestry University, Supervisor: Pan D M, pp. 87-88.) [5] 李宪利, 袁志友, 高东升. 2002. 高等植物成花分子机理研究现状及展望[J]. 西北植物学报, 22(1): 173-183. (Li X L, Yuan Z Y, Gao D S.2002. The research achievements on mechanism of floral formation in plants[J]. Acta Botanica Boreali-Occidentalia Sinica, 22(1): 173-183.) [6] 李小方, 王洋, 邓新杰, 等. 2009. 温度、GA3和乙烯对中国水仙休眠的解除[J]. 植物生理学报, 45(10): 953-957. (Li X F, Wang Y, Deng X J, et al.2009. Release of temperature, GA3 and ethylene to the dormancy of Chinese narcissus(Narcissus tazetta var. chinensis Roem.)[J]. Plant Physiology Journal, 45(10): 953-957.) [7] 李兴军, 李三玉, 吕均良, 等. 2001. GA3对杨梅叶片木质素水平及其相关酶活性和成花的影响[J]. 园艺学报, 28(2): 156-158. (Li X J, Li S Y, Lü J L, et al.2001. Effects of Gibberellin3 on leaf lignin levels, related enzymes and flower formation in bayberry[J]. Acta Horticulturae Sinica, 28(2): 156-158.) [8] 林葵, 黄祥辉, 王隆华, 等. 1996. 甜瓜子叶不定芽分化过程中PAL活性和木质素含量变化研究[J]. 华东师范大学学报(自然科学版), (2): 92-97. (Lin K, Huang X H, Wang L H, et al. 1996. The change of PAL activity and lignin content during adventitious buds formation of cotyledon in Cucumis melon L[J]. Journal of East China Normal University, (2): 92-97.) [9] 徐东花, 郭俊娥, 徐璐, 等. 2014. 多胺氧化酶活性及木质素沉积与菊花花芽分化的关系[J]. 华北农学报, 29(3): 164-169. (Xu D H, Guo J E, Xu L, et al.2014. The relationship between polyamine oxidase activity and lignin deposition and chrysanthemum flower bud differentiation[J]. Acta Agriculturae Boreali-Sinica, 29(3): 164-169.) [10] 王学奎. 2006. 植物生理生化实验原理和技术[M]. 高等教育出版社, 北京. pp. 167-204. (Wang X K.2006. Principles and Techniques of Plant Physiological Biochemical Experiment[M]. Higher Education Press, Beijing. pp. 167-204.) [11] 武丹, 吴菁华, 张志忠. 2017. 中国水仙NtSOC1基因克隆及亚细胞定位[J]. 西北植物学报, 37(10): 1889-1895. (Wu D, Wu J H, Zhang Z Z.2007. Cloning and sub-cellular localization of NtSOC1 gene from Narcissus tazetta var. chinensis[J]. Acta Botanica Boreali-Occidentalia Sinica, 37(10): 1889-1895.) [12] 杨超, 吴菁华, 吴少华. 2012. 中国水仙NtAP1基因的克隆与表达分析[J]. 西北植物学报, 34(6): 1084-1088. (Yang C, Wu J H, Wu S H.2012. Clone and expression of NtAP1 honologous gene from Narcissus tazetta var. chinensis[J]. Acta Botanica Boreali-Occidentalia Sinica, 34(6): 1084-1088.) [13] 叶季波. 2009. 中国水仙鳞茎催花技术机理及应用研究[J]. 中国园艺文摘, 25(7): 26-28. (Ye J B.2009. Mechanism and application of flower promoting technology of Narcissus tazetta var. chinese[J]. Chinese Horticulture Abstracts, 25(7): 26-28.) [14] 张乔松, 杨伟儿. 1987. 中国水仙花芽分化及贮藏期外界因子对花序数的影响[J]. 园艺学报, 14(2): 139-144. (Zhang Q S, Yang W E.1987. On flower-bud differentiation of Chinese narcissus and the effect of external factors in storage on flower percentage[J]. Acta Horticulturae Sinica, 14(2): 139-144.) [15] Abeles F B.1967. Inhibition of flowering in Xanthium pensylvanicum Walln. by ethylene[J]. Plant Physiology, 42(4): 608-609. [16] Audic S, Claverie J M.1997. The significance of digital gene expression profiles[J]. Genome research h, 7(10): 986-995. [17] Brady S M, Song S, Dhugga K S, et al.2007. Combining expression and comparative evolutionary analysis the COBRA gene family[J]. Plant Physiology, 143(1): 172-187. [18] Feng Y, Zhu L, Pan T, et al.2015. Characterization of summer dormancy in Narcissus tazetta var. chinensis and the role of NtFTs in summer dormancy and flower differentiation[J]. Scientia Horticulturae, 183: 109-117. [19] Gao J, Liu W F.2008. Isolation and characterization of the LEAFY homologus gene NTLEAFY in Chinese narcissus (Narcissus tazetta var. chinensis Roem)[J]. Journal of Biotechnology, 136(4): S223-S223. [20] Grasmanis V O, Leeper G W.1967. Ammonium nutrition and flowering of apple trees[J]. Australian Journal of Biological Sciences, 20(4): 761-768. [21] Groppa M D, Benavides M P.2008. Polyamines and abiotic stress: Recent advances[J]. Amino Acids, 34(1): 35. [22] Han S S, Halevy A H, Sachs R M, et al.1990. Enhancement of growth and flowering of Triteleia laxa by ethylene[J]. Journal of the American Society for Horticultural Science,115(3): 482-486. [23] Imanishi H.1983. Effects of exposure of bulbs to smoke and ethylene on flowering of Narcissus tazetta cultivar ‘Grand Soleil d 'Or’[J]. Scientia Horticulturae, 21(2): 173-180. [24] Kim D H, Doyle M R, Sung S, et al.2009. Vernalization: Winter and the timing of flowering in plants[J]. Annual Review of Cell and Developmental Biology, 25(1): 277-299. [25] Kusano T, Berberich T, Tateda C, et al.2008. Polyamines: Essential factors for growth and survival[J]. Planta, 228(3): 367-381. [26] Lavee S.1989. Involvement of plant growth regulators and endogenous growth substances in the control of alternate bearing[J]. Acta Horticulturae, 239(1): 311-322. [27] Li X F, Jia L Y, Xu J, et al.2013. FT-like NFT1 gene may play a role in flower transition induced by heat accumulation in Narcissus tazetta var. chinensis[J]. Plant and Cell Physiology, 54(2): 270-281. [28] Li X F, Shao X H, Deng X J, et al.2012. Necessity of high temperature for the dormancy release of Narcissus tazetta var. chinensis[J]. Journal of Plant Physiology, 169(14): 1340-1347. [29] Moerschbacher B, Heck B, Kogel K H, et al.1986. An elicitor of the hypersensitive lignification response in wheat leaves isolated from the rust fungus Pucciia graminis f. sp. Tritici II. induction of enzymes corre lated with biosynthesis of lignin[J]. Zeitschrift Fur Naturorsch, 41(9): 839-844. [30] Morita S, Murakoshi Y, Ai H, et al.2012. Early flowering and increased expression of a FLOWERING LOCUS T-like, gene in chrysanthemum transformed with a mutated ethylene receptor gene mDG-ERS1(etr1-4)[J]. Journal of Plant Biology, 55(5): 398-405. [31] Mortazavi A, Williams B A, McCue K, et al.2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nature Methods, 5(7): 621-628. [32] Qiu J D, Luo X Z, Wu D Y.2001. Regulation of flower bud differentiation in longan[J]. Acta Horticulturae, 558: 225-228. [33] Rossi V, Locatelli S, Varotto S, et al.2007. Maize histone deacetylase hda101 is involved in plant development, gene transcription, and sequence-specific modulation of histone modification of genes and repeats[J]. Plant Cell, 19(4): 1145-1162. [34] Tian L, Chen Z J.2001. Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development[J]. Proceedings of the National Academy of Sciences of the USA, 98(1): 200-205. [35] Zhang D Q, Yang X H, Zhang Z Y, et al.2010. Expression and nucleotide diversity of the poplar COBL gene[J]. Tree Genetics and Genomes, 6(2): 331-344.
