Transcriptome and Metabolome Reveal Salt-Stress Responses of Leaf Tissues from Dendrobium officinale

Abstract

Dendrobium officinale Kimura et Migo is a precious traditional Chinese medicine. Despite D. officinale displaying a good salt-tolerance level, the yield and growth of D. officinale were impaired drastically by the increasing soil secondary salinization. The molecular mechanisms of D. officinale plants' adaptation to salt stress are not well documented. Therefore, in the present study, D. officinale plants were treated with 250 mM NaCl. Transcriptome analysis showed that salt stress significantly altered various metabolic pathways, including phenylalanine metabolism, flavonoid biosynthesis, and α-linolenic acid metabolism, and significantly upregulated the mRNA expression levels of DoAOC, DoAOS, DoLOX2S, DoMFP, and DoOPR involved in the jasmonic acid (JA) biosynthesis pathway, as well as rutin synthesis genes involved in the flavonoid synthesis pathway. In addition, metabolomics analysis showed that salt stress induced the accumulation of some compounds in D. officinale leaves, especially flavonoids, sugars, and alkaloids, which may play an important role in salt-stress responses of leaf tissues from D. officinale. Moreover, salt stress could trigger JA biosynthesis, and JA may act as a signal molecule that promotes flavonoid biosynthesis in D. officinale leaves. To sum up, D. officinale plants adapted to salt stress by enhancing the biosynthesis of secondary metabolites.

Keywords: Dendrobium officinale; flavonoids; jasmonic acid; metabolome; salt stress; transcriptome.

Figure 1

Differentially expressed genes (DEGs) at different time points under salt treatments. (A) Number of DEGs. (B) Venn diagram of DEGs. (C) Top 20 enriched pathways for overlapping salt responsive genes of 0 h vs. 4 h, 0 h vs. 12 h, and 4 h vs. 12 h.

Figure 2

qRT-PCR confirmation of expression profiles obtained by RNA-Seq transcriptome analysis at different times after salt treatment. Fold-change in transcript abundance obtained by both qRT-PCR and RNA-Seq are presented on the same graph for eight representative encoding genes. Column (A) shows the fold-change in the Fragments Per Kilobase of transcript sequence per Millions mapped reads (FPKM) value obtained by RNA-Seq, and column (B) shows the fold-change in the expression determined by qRT-PCR. DoANS, DoAOC, DoAOS, DoCHI, DoF3′5′H, DoLOX2S, DoMFP, and DoOPR are anthocyanin synthase, allene oxide cyclase, allene oxide synthase, chalcone isomerase, flavonoid 3′,5′-hydroxylase, lipoxygenase, peroxisomal fatty acid beta-oxidation multifunctional protein, and 12-oxophytodienoate reductase, respectively, in D. officinale. Data represent means and SDs. Statistical significance was calculated using GraphPad Prism 8 (Version 8.0.2, GraphPad Software, La Jolla, CA, USA). * p < 0.05; ** p < 0.01.

Figure 3

Heat map analysis of the overall gene expression profiles and Kyoto Encyclopedia of Genes and Genomes (KEGG) category distribution of the DEGs in the six expression subclusters composing the transcriptome of D. officinale leaves under salt stress. (A) The hierarchical analyses of co-expression transcripts at different time points of salt stress. Blue indicates the downregulated and red indicates the upregulated expression of genes. Expression values were Z-scaled (log₂(FPKM+1)). (B) Six subclusters of expression patterns and KEGG enrichment categories. Expression values were Z-scaled. The blue line represents the average expression value of the members at each time point. The degree of enrichment of categories in each cluster is shown in different colored boxes. Red represents the over-represented categories and blue represents the under-represented categories. KEGG category enrichment was computed using the Cluster package and q value package (q value < 0.05) in R.

Figure 4

The jasmonic acid (JA) biosynthetic pathway and related upregulated genes under salt treatment. (A) The JA biosynthetic pathway and its associated enzymes. (B) Heat map analysis of upregulated gene expression in the JA biosynthetic pathway under salt treatment.

Figure 5

Sugar, amino acid, and flavonoid metabolic pathways and related upregulated genes under salt stress. (A) Sugar, amino acids, and flavonoid metabolic pathways. Blue text shows the value of log₂fold change (FC) in metabolites under salt stress (see Table S5 for details). (B) Heat map analysis of upregulated gene expression under salt stress.

Figure 6

The ascorbic acid biosynthesis pathway and related upregulated genes under salt treatment. (A) The ascorbic acid biosynthesis pathway. (B) Heat map analysis of upregulated gene expression under salt treatment.

Figure 7

Heat map analysis of upregulated gene expression associated with the ascorbate-glutathione (AsA-GSH) cycle under salt treatment.

Figure 8

The contents of JA, total flavonoids, and anthocyanidin in leaves under salt stress. (A) JA content at different time points under salt stress. (B) The contents of total flavonoids in control and salt-stress treatments. (C) The contents of anthocyanidin in control and salt-stress treatments. Statistical significance was calculated using Student’s t-tests. * p < 0.05, ** p < 0.01.