Physiological Response and Transcriptome Analysis of Tobacco Yunongxiang 201 to Low Temperature Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-0579

Abstract

Abstract:

Objective Yunongxiang 201 is a new flue-cured tobacco variety by self-crossing six generations, which was selected from the Zhongyan 100 0.6% EMS mutant library, with good resistance diseases and elite tobacco quality, whereas, it in field showed low-temperature sensitivity and early flowering characteristics. The molecular mechanism formed by low-temperature sensitivity of Yunongxiang 201 was studied. Method Yunongxiang 201 and Zhongyan 100 were used as test materials, their growth status was observed under 4 ℃ stress for 0 h, 6 h and 24 h. The physiological indexes of H₂O₂ content, proline content, antioxidant enzyme activity and endogenous ABA content was determined, and the growth status of plants recovered at 25 ℃ for 15 d was observed. The molecular mechanism was explored by combining transcriptomics. Result Compared with Zhongyan 100, Yunongxiang 201 wilted seriously at 4 ℃ for 24 h. Encountering low temperature, the H₂O₂ content, proline content, SOD and POD antioxidant enzymes in Zhongyan 100 increased significantly, and the content of endogenous ABA increased significantly under continuous low temperature; interestingly, the content of H₂O₂ in Yunongxiang 201 increased significantly, the content of proline, SOD and POD antioxidant enzymes didn’t increase significantly, and the content of endogenous ABA decreased significantly. The number of differential expressed genes (DEGs) involved in low- temperature-response of Zhongyan 100 and Yunongxiang 201 respectively were 4 314 and 3 769 at 6 h; Zhongyan 100 and Yunongxiang 201 respectively were 17 618 and 14 293 at 24 h. KEGG enrichment showed that the number of DEGs involved in starch-sucrose metabolism pathway was similar in the two varieties, Zhongyan 100's DEGs were more involved in plant hormone transduction pathway, and Yunongxiang 201’s DEGs were more involved in plant-circadian rhythm pathway under cold stress. After encountering low temperature, the expressions of soluble sugar related genes SUS, TPP, α-amylasegene AMY3, as well as ABA pathway-related genes SRK2E, SAPK3 were up-regulated in Zhongyan 100, which was conducive to enhancing cold resistance. The expressions of flowering pathway-related genes ELF3, FLK, cold-regulating gene COR27, constitutive photomorphogenic1 gene COP1 and CBF negative regulator EIN3 in Yunongxiang 201 was up-regulated. Conclusion After low temperature, the broad and complex cold-resistant defense pathway of Zhongyan 100 is activated, while the antioxidant enzyme system is insufficiently regulated, thus the ROS scavenging system were weakened, resulting in the more sensitive of Yunongxiang 201 to low temperature.

Key words: Nicotiana tabacum, cold stress, Yunongxiang 201, transcriptomics, physiological response

WANG Ya-le, LI Xue-jun, SUN Ji-ping, SUN Huan. Physiological Response and Transcriptome Analysis of Tobacco Yunongxiang 201 to Low Temperature Stress[J]. Biotechnology Bulletin, 2026, 42(2): 228-238.

Figures/Tables 9

Table 1 Primer sequences detected by RT-qPCR

基因名称 Name	上游引物序列 Forward primer sequences（5′-3′）	下游引物序列 Reverse primer sequences（5′-3′）
NtFLK	TAGGGACGGTGCTCAATTCAA	GGACATTACCAGCGTCTTGC
NtEIN3	AGCCTCGTCAACAACCTAGC	TGTTGGTGTCCCCGAAGATG
NtSUS	AGTTCACCACTACAAGGGCAAGA	AGGGGAAAGGGTAGTGAGGTATT
NtTPP	GGATTCGGATGCTAAAGCGG	TACTTGTGATGAGTTGGAGATGA
NtTIFY10A	TGGAAACCAAACAACTACTACTATG	GTCATTTGTGCCTTTTCTGGTT
NtSAPK3	GAGGTCTTGGTAACTCCATCG	CACATATTTTTACACGTGGTGAAGC
NtCOP1	ACCTGGACAGGCAATAGCAAG	TCTTCAAGTCCCGCAGAATAAC
NtbHLH13	CGAGGATGGGGCTCAACAA	ATAACCACAAATGCTTACCCG
L25	CCCCTCACCACAGAGTCTGC	AAGGGTGTTGTTGTCCTCAATCTT

Fig. 1 Different tolerance of Yunongxiang 201 and Zhongyan 100 to cold stressA-C: The phenotypes of 0 h (A), 6 h (B) and 24 h (C) at 4 ℃ in Yunongxiang 201 and Zhongyan 100. Bar=2 cm. D: Physiological index of Yunongxiang 201 and Zhongyan 100 before and after 4 ℃ treatment. Different uppercase letters indicate significant differences (P<0.01), different lowercase letters indicate significant differences (P＜0.05)

Fig. 2 Volcano and Venn maps of DEGs

Fig. 3 KEGG enrichment analysis of DEGs among different groups

Fig. 4 Expression analysis of DEGs in plant hormone pathway in Yunongxiang 201 and Zhongyan 100A: Statistics of DEGs in plant hormone pathway at different time points. B: ABA content in leaves at different time points. C: The map of KEGG in plant hormone pathway. Red indicates up-regulated expression gene, green indicates down-regulated expression gene, and blue indicates partial genes up or down-regulated expression at 0 h_vs_6 h. The left side indicates Yunongxiang 201(YN) and the right side indicates Zhongyan 100(ZY) at each node in plant hormone pathway

Fig. 5 Expression analysis of enzyme genes related to starch-sucrose metabolic pathways in Yunongxiang 201 and Zhongyan 100A: Statistics of DEGs at different time points. B: Expression heat map of related enzyme genes in starch-sucrose metabolic pathway. Asterisk indicates FDR<0.05 in heat map, the same below

Fig. 6 Enrichment of DEGs in circadian rhythm-plant pathway in Yunongxiang 201 and Zhongyan 100

Fig. 7 Verification of DEGs by RT-qPCR

Fig. 8 Part of the gene regulatory network involved in cold tolerance and flowering process

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