Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation

Abstract

Plant cold acclimation (CA) is a genetically complex phenomenon involving gene regulation and expression. Little is known about the cascading pattern of gene regulatroy network and the link between genes and metabolites during CA. Dendrobium officinale (DOKM) is an important medicinal and ornamental plant and hypersensitive to low temperature. Here, we used the large scale metabolomic and transcriptomic technologies to reveal the response to CA in DOKM seedlings based on the physiological profile analyses. Lowering temperature from 4 to -2°C resulted in significant increase (P < 0.01) in antioxidant activities and electrolyte leakage (EL) during 24 h. The fitness CA piont of 0°C and control (20°C) during 20 h were firstly obtained according to physiological analyses. Subsequently, massive transcriptome and metabolome reprogramming occurred during CA. The gene to metabolite network demonstrated that the CA associated processes are highly energy demanding through activating hydrolysis of sugars, amino acids catabolism and citrate cycle. The expression levels of 2,767 genes were significantly affected by CA, including 153-fold upregulation of CBF transcription factor, 56-fold upregulation of MAPKKK16 protein kinase. Moreover, the gene interaction and regulation network analysis revealed that the CA as an active process, was regulated at the transcriptional, post-transcriptional, translational and post-translational levels. Our findings highligted a comprehensive regulatory mechanism including cold signal transduction, transcriptional regulation, and gene expression, which contributes a deeper understanding of the highly complex regulatory program during CA in DOKM. Some marker genes identified in DOKM seedlings will allow us to understand the role of each individual during CA by further functional analyses.

Keywords: Cold acclimation; Dendrobium officinale; metabolic network; regulatory mechanism; signal transduction; transcriptome.

FIGURE 1

Antioxidant enzymes responses of Dendrobium officinale to cold acclimation. The activities of (A) superoxide dismutase (SOD) activity, (B) catalase (CAT) activity, (C) peroxidase (POD) activity, and (D) ascorbate peroxidase (APX) were quantified by the means of three independent experiments ± SD. Different letters mark indicates significantly different at P < 0.01.

FIGURE 2

Cold tolerance expressed as electrolyte leakage of D. officinale under cold acclimation and control. Results are shown as means ± SD from three independent experiments. Different letters mark indicates significantly different at P < 0.01.

FIGURE 3

Metabolome reprogramming during cold acclimation (CA) in comparison with control. Shown are the metabolome profiles for ten independent biological replicates of CA and parallel control replicates (CK). (A) Principal component analysis (PCA) depicted a metabolome reprogramming during CA as compared with control. Values for x and y axes are scores for PC1 and PC2. (B) Hierarchical cluster analysis (HCA) of all differentially accumulated metabolites between CA and control treatments. The bar at bottom represents the color code for log₂-transformed data of metabolite accumulation using the CLUSTER software package. The levels of metabolite accumulation are listed in Supplementary Table S1 in Supplementary Material.

FIGURE 4

Global expression profiles and interaction network of differentially expressed genes (DEGs) during cold acclimation (CA) as compared with control growth. (A) HCA, indicating that all DEGs were grouped into five clusters (C1–C5) based on their expression levels. The numbers at the left of the map represent the amount of DEGs in each cluster. The red and green arrows mean up-regulation and down-regulation. (B) Interaction networks depicted multiple hubs (C1–C6) of gene interaction under CA condition. The network was generated based on the potential interaction data for 2,767 DEGs, which were extracted from the latest STRING database (STRING v10). Node size indicates its connectivity measured as node degree (i.e., the number of edges connecting the node); the bigger node means a higher connectivity. The edge (the connecting line) indicates the interaction between two genes, and the weight of edges between them is a measure of their interaction. Thick edges show strong interaction, whereas thin edges show weak interaction. The bar at bottom shows the color code for the fold change in gene expression of log₂ -transformed data. All interaction data are available in Supplementary table S5 in Supplementary Material.

FIGURE 5

The energy homeostasis strategies through carbohydrate and amino acid metabolic pathways, indicating massive transcriptome and metabolome reprogramming during cold acclimation (CA). The magenta and cyan squares represent up-regulation and down-regulation of a biosynthetic gene. The metabolites in magenta represent up-regulated accumulation under CA compared with control. The metabolites in bold show no differences in accumulation, and the metabolites in gray are not identified by GC-MS. ACO, aconitate hydratase; AlAT, alanine aminotransferase; ANIN, alkaline and neutral invertase; AST, aspartate aminotransferase; CIN, cell wall invertase; FBA, fructose-bisphosphate aldolase; Glu-6-P, glucose-6-phosphate; G3DP, glyceraldehyde 3-phosphate dehydrogenase; G3P, glyceraldehyde-3-phosphate; HK, hexokinase; ICL, isocitrate lyase; MDH, malate dehydrogenase; PEP, phosphoenol pyruvate; 3-PGA, 3-phosphoglycerate; PGDH, D-3-phosphoglycerate dehydrogenase; PGM, phosphoglycerate mutase; PPDK, pyruvate phosphate dikinase; PSAT, phosphoserine aminotransferase; SDH, succinate dehydrogenase.

FIGURE 6

Model of multiple signal transduction pathways and transcriptional networks during cold acclimation in D. officinale. Red lines represent Ca²⁺-dependent signaling process and CBF-dependent regulatory pathway; green lines represent Ca²⁺-independent signaling process and ABA-dependent regulator pathway. Arrows represent positive regulation, whereas lines ending with a bar represent negative regulation. The three stars (^∗∗∗) indicate unknown cis-elements. ABF, ABA-responsive element binding factor; ABO5, ABA overly- ensitive 5; ABRE, ABF recognition element; ASR1, ABA stress-ripening protein 1; CaM, calmodulin; CBL, calcineurin B-like Ca²⁺ sensors; CDPK, Ca²⁺-dependent protein kinase; COR27/47, cold regulated gene 27/47; CRT, C-repeat elements; DRE, dehydration-responsive elements; ERD4, dehydration-induced protein 4; FADs, fatty acid desaturases; GPCR, G-protein-coupled receptor; HSPs, heat shock proteins; LEAs, late embryogenesis abundant protein; LTPs, lipid-transfer proteins; MYBRS, MYB transcription factor recognition sequence; PEIs, pectinesterase inhibitors; PP2C, 2C-type protein phosphatase; PRPs, proline-rich proteins; PYL, pyrabactin resistance 1-like; RAB18, dehydrin family protein 18; RAS, rat sarcoma protein; RTKs, receptor-tyrosine kinases; SnRK2, SNF1-related protein kinase 2; P, phosphorylation; S, sumoylation; U, ubiquitination.