Abstract
Isatis indigotica Fort., as a common Chinese medicinal raw material, will lose its medicinal value if it blooms early, so it is highly valuable to clarify the induction mechanism of the vernalization of I. indigotica at low temperature. In this study, the concentrations of soluble sugar, proline, glutathione and zeatin in two germplasms of I. indigotica with different degrees of low temperature tolerance (Y1 and Y2) were determined at 10 days, 20 days and 30 days of low-temperature treatment, and the full-length transcriptome of 24 samples was sequenced by Nanopore sequencing with Oxford Nanopore Technologies (ONT). After that, the data of transcripts involved in the vernalization of I. indigotica at low temperature were obtained, and these transcripts were identified using weighted gene co-expression network analysis (WGCNA). The results revealed the massive accumulation of soluble sugar and proline in Y1 and Y2 after low temperature induction. A total of 18,385 new transcripts, 6168 transcription factors and 470 lncRNAs were obtained. Differential expression analysis showed that gibberellin, flavonoids, fatty acids and some processes related to low temperature response were significantly enriched. Eight key transcripts were identified by WGCNA, among which ONT.14640.1, ONT.9119.1, ONT.13080.2 and ONT.16007.1 encodes a flavonoid transporter, 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), growth factor gene and L-aspartate oxidase in plants, respectively. It indicated that secondary metabolites such as hormones and flavonoids play an important role in the vernalization of I. indigotica. qRT-PCR proved the reliability of transcriptome results. These results provide important insights on the low-temperature vernalization of I. indigotica, and provide a research basis for analyzing the vernalization mechanism of I. indigotica.
Highlights
Isatis indigotica Fort., as a common Chinese medicinal raw material, will lose its medicinal value if it blooms early, so it is highly valuable to clarify the induction mechanism of the vernalization of I. indigotica at low temperature
Using weighted gene co-expression network analysis (WGCNA), it has been verified that ethylene, jasmonic acid, zinc finger-containing transcription factors (TFs) and auxin are involved during vernalization in A. thaliana acclimated at low temperature (Li et al 2021)
The concentrations of soluble sugar, proline, glutathione and zeatin in the seedlings of two germplasms of I. indigotica, namely, Y1 and Y2, at low temperature were determined at 0 days, 10 days, 20 days and 30 days, respectively
Summary
Isatis indigotica Fort., as a common Chinese medicinal raw material, will lose its medicinal value if it blooms early, so it is highly valuable to clarify the induction mechanism of the vernalization of I. indigotica at low temperature. Eight key transcripts were identified by WGCNA, among which ONT.14640.1, ONT.9119.1, ONT.13080.2 and ONT.16007.1 encodes a flavonoid transporter, 9-cisepoxycarotenoid dioxygenase 3 (NCED3), growth factor gene and L-aspartate oxidase in plants, respectively It indicated that secondary metabolites such as hormones and flavonoids play an important role in the vernalization of I. indigotica. Weighted Gene Co-Expression Network Analysis (WGCNA) takes advantage of the interrelated characteristics of various life activities in plants to obtain many expression characteristics of life activities using highthroughput sequencing technology and calculates the correlation between genes to construct the scale-free network topology, that is, it links most genes with regulatory relationships with key regulatory genes as the core (Zhang and Horvath 2005) In most cases, this method is applied to identify the biological relationship between genes related to plant traits, and it plays a role in the identification of key regulatory genes under the low-temperature induction in plants. Using WGCNA, it has been verified that ethylene, jasmonic acid, zinc finger-containing transcription factors (TFs) and auxin are involved during vernalization in A. thaliana acclimated at low temperature (Li et al 2021). Li et al (2020) identified that a gene CpFT1 acclimated at low temperature in Chimonanthus praecox using WGCNA, and found that its overexpression in A. thaliana can promote flowering
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