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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2023, Vol. 49 Issue (3): 305-318    DOI: 10.3785/j.issn.1008-9209.2022.05.311
Crop sciences     
Physiological response and its transcriptomic and secondary metabolic characteristics oftobacco seedlings under well-cellar style transplanting
Yechun LIN1(),Wenjie TONG2,Dairong CHEN3,Lingjie XU4,Shixiang JIANG5,Youxiang WU6,Yang YANG6,Yongfeng AI3,Hongxun LI1,Wenjie PAN4()
1.Upland Flue-cured Tobacco Quality & Ecology Key Laboratory of China Tobacco, Guizhou Academy of Tobacco Science, Guiyang 550081, Guizhou, China
2.Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, Yunnan, China
3.Tongren Branch Company of Guizhou Provincial Tobacco Company, Tongren 554300, Guizhou, China
4.Guizhou Tobacco Company, China National Tobacco Corporation, Guiyang 550000, Guizhou, China
5.Zunyi Branch Company of Guizhou Provincial Tobacco Company, Zunyi 563000, Guizhou, China
6.China Tobacco Guizhou Industrial Co. , Ltd. , Guiyang 550009, Guizhou, China
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Abstract  

In order to demonstrate the mechanism of promoting crop growth by the well-cellar style transplanting (WCST), this study analyzed the physiological, biochemical and transcriptomic characteristics of tobacco seedlings under WCST compared with the conventional transplanting (CT), in which seedlings were transplanted on membranes. The results showed that under WCST, the average air temperature and average relative humidity of the atmosphere were 22.29 ℃ and 97.10%, enhanced by 4.76 ℃ and 58.97% compared with CT, respectively. However, the average photosynthetically active radiation (PAR) was 221.57 μmol/(m2·s), reduced by 20.53% than CT. After transplanting, the plant height and maximum leaf area under WCST were significantly improved, and the leaf water potential was enhanced between 29.92% and 64.46%. Furthermore, superoxide dismutase (SOD), peroxidase (POD) activities and malonaldehyde (MDA) content were reduced by 14.48%, 8.86%, and 9.83% compared with CT (P＜0.05), respectively. There were 4 845 differentially expressed genes (DEGs) identified, including 2 693 down-regulated genes and 2 152 up-regulated genes according to transcriptome sequencing. Some down-regulated DEGs are significantly enriched in “oxidation-reduction process” and “oxidoreductase activity” according to gene ontology (GO) annotation. Some DEGs involved in “flavonoid biosynthesis” are significantly enriched according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. WCST decreased the gene expressions of chalcone isomerase, flavanone-3-hydroxylase,flavonol synthase,flavonoid-3′-hydroxylase,andflavonol-3-O-glucoside L-rhamnosyltransferaserelated with flavonoid biosynthesis, and significantly reduced the contents of the dihydroquercetin, rutin, kaempferol-3-O-glucoside, quercetin-3-O-galactoside, luteolin, isorhamnetin-3-O-rutinoside and kaempferol-3-O-rutinoside. The higher air temperature, air relative humidity and lower PAR were beneficial to the growth and development of tobacco seedlings under WCST, which avoiding the adverse stress.

Key words： conventional transplanting      well-cellar style transplanting      adverse stress      RNA sequencing      flavonoid biosynthesis     
Received: 31 May 2022      Published: 25 June 2023
CLC:  S572  
Corresponding Authors: Wenjie PAN     E-mail: linyechun@live.cn;wenjiepan@163.com
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Yechun LIN
Wenjie TONG
Dairong CHEN
Lingjie XU
Shixiang JIANG
Youxiang WU
Yang YANG
Yongfeng AI
Hongxun LI
Wenjie PAN
Cite this article:

Yechun LIN,Wenjie TONG,Dairong CHEN,Lingjie XU,Shixiang JIANG,Youxiang WU,Yang YANG,Yongfeng AI,Hongxun LI,Wenjie PAN. Physiological response and its transcriptomic and secondary metabolic characteristics oftobacco seedlings under well-cellar style transplanting. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 305-318.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.05.311     OR     https://www.zjujournals.com/agr/Y2023/V49/I3/305


井窖深栽烟苗的生理响应及其转录组和次生代谢特征

为揭示井窖深栽促进作物生长的理论机制，以常规移栽为对照，比较分析了井窖深栽烟苗的生理生化和转录组特征。结果表明：井窖深栽烟苗生长环境的平均气温为22.29 ℃，比常规移栽提高了4.76 ℃；环境相对湿度为97.10%，较常规移栽增加了58.97%；烟苗叶片的平均太阳光合有效辐射为221.57 μmol/（m2·s），比常规移栽降低了20.53%。与常规移栽相比，井窖深栽烟苗株高显著增加，最大叶面积显著增大，叶片水势提高29.92%～64.46%，增加显著；叶片超氧化物歧化酶（superoxide dismutase, SOD）、过氧化物酶（peroxidase, POD）活性及丙二醛（malonaldehyde, MDA）含量分别降低了14.48%、8.86%和9.83%（P＜0.05）。转录组测序共发现4 845个差异表达基因（differentially expressed genes, DEGs），其中下调表达基因2 693个，上调表达基因2 152个；基因本体（gene ontology, GO）富集分析表明，下调DEGs主要富集于氧化还原过程和氧化还原酶活性等功能途径；京都基因和基因组数据库（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析表明，DEGs最显著富集于黄酮类化合物生物合成途径，该途径关键酶查耳酮异构酶、黄烷酮-3-羟化酶、黄酮醇合成酶、黄酮类-3′-羟化酶、黄酮醇-3-O-葡萄糖苷L-鼠李素转移酶等的相关基因表达下调。常规移栽烟苗叶片中的二氢槲皮素、芦丁、山柰酚-3-O-葡萄糖苷、槲皮素-3-O-半乳糖苷、木犀草素、异鼠李素-3-O-芸香糖苷和山柰酚-3-O-芸香糖苷等7种黄酮类化合物含量显著高于井窖深栽烟苗。井窖深栽提高了移栽烟苗生长环境的温、湿度，降低了叶片光合有效辐射，避免了烟苗遭遇明显逆境胁迫，促进了烟苗的生长发育。


关键词： 常规移栽,  井窖深栽,  逆境胁迫,  RNA测序,  黄酮类生物合成 
Fig. 1 Two transplanting methods of tobacco seedlings in the field and pot experiments

序号

No.

基因

Gene

正向引物（5′→3′）

Forward primer (5′→3′)

反向引物（5′→3′）

Reverse primer (5′→3′)

1gene_71163TGAAGGCCTTGATTGGGGTGAAGCCAATACCCACACATGAAC
2gene_23271AGACACCTAGCATCCATTTTCCTTGGCCAACCTCTTGGTGTTT
3gene_45893CCTCTCGCTCCACCTAACCTTTGCTGGTGGGAACACGTAA
4gene_78573AGAAGGCCAAGTCGGAAACCCACCAATTCAAAACGGCAAGA
5gene_57394AAGTAGCGCTGATCCACAGGGGTCAAGAACGCGTCGAAAC
6gene_42505AGCACGTGAATACTAGCTACCAGCTCCTCGACGGTGACCATTT
7gene_78071TTTCCGGCGAAAATGGTGACAGTTGGAAGGTGTGGCTGTT
8gene_2555GTTTCGACGCGTTCTTGACCTGCCGATTCAGGTGCGTATC
9GAPDH2CCGTAAGACTAGAGAAAGAAGCCTGCCTTGGCATCGAAAATGCT
Table 1 Genes and primers for qRT-PCR
Fig. 2 Air temperatures for tobacco seedling growth environment under different transplanting methods (2017)
Fig. 3 Relative humidities for tobacco seedling growth environment under different transplanting methods (2017)
Fig. 4 Photosynthetically active radiation for tobacco seedling growth environment under different transplanting methods (2017)
Fig. 5 Physiological characteristics and agronomic traits of tobacco seedlings under different transplanting methods (2017)Single asterisk (*) indicate significant differences between different transplanting methods on the same date at the 0.05 probability level.
Fig. 6 Volcano plot of expression levels of DEGs in tobacco seedling leaves under WCST and CTRed dots: Up-regulated genes (2 152); Blue dots: Down-regulated genes (2 693). FC: Fold change.
Fig. 7 GO function annotation analysis of the DEGsA. Biological process; B. Molecular function; C. Cellular component. Value on the right side of histogram is the number of DEGs. Single asterisk (*), double asterisks (**), and triple asterisks (***) indicate significant differences between different gene expressions of the same function at the 0.05, 0.01, and 0.001 probability levels, respectively.
Fig. 8 KEGG enrichment scatter diagram of the DEGs
Fig. 9 Biosynthesis pathways of some flavonoidsThe related genes of the key enzymes marked in green are down-regulated, and the flavonoids marked in red represent the determined flavonoids. PAL: Phenylalanine ammonia-lyase; C4H: Cinnamate-4-hydroxylase; 4CL: 4-coumaroyl-CoA ligase; HCT: Hydroxycinnamoyl transferase; C3H: p-coumarate-3-hydroxylase; CHS: Chalcone synthase; CHI: Chalcone isomerase; F3H: Flavanone-3-hydroxylase; F3´H: Flavonoid-3´-hydroxylase; FLS: Flavonol synthase; F3´5´H: Flavonoid-3´, 5´-hydroxylase; FNS: Flavone synthase; F3GT: Flavonol-3-O-glucosyltransferase; FG2: Flavonol-3-O-glucoside L-rhamnosyltransferase.
 
Fig. 11 Contents of the flavonoids in tobacco seedling leaves under different transplanting methodsSingle asterisk (*) indicate significant differences at the 0.05 probability level.
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