Physiological and transcriptomic comparisons shed light on the cold stress response mechanisms of Dendrobium spp

Abstract

Background: Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold response regulatory mechanisms in this group are unclear. To understand how these plants respond to cold stress, we compared the transcriptomic responses of the cold-tolerant cultivar 'Hongxing' (HX) and the cold-sensitive cultivar 'Sonia Hiasakul' (SH) to cold stress.

Results: Chemometric results showed that the physiological response of SH in the later stages of cold stress is similar to that of HX throughout the cold treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that soluble protein content and peroxidase activity are key physiological parameters for assessing the cold tolerance of these two Dendrobium spp. cultivars. Additionally, weighted gene co-expression network analysis (WGCNA) results showed that many cold response genes and metabolic pathways significantly associated with the physiological indices were enriched in the 12 detected modules. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses of the 105 hub genes showed that Dendrobium spp. adapt to cold stress by regulating signal transduction, phytohormones, transcription factors, protein translation and modification, functional proteins, biosynthesis and metabolism, cell structure, light, and the circadian clock. Hub genes of the cold stress response network included the remorin gene pp34, the abscisic acid signaling pathway-related genes PROTEIN PHOSPATASE 2 C (PP2C), SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ABRE-BINDING FACTOR 1 (ABF1) and SKI-INTERACTING PROTEIN 17 (SKIP17), the Ca²⁺ signaling-related GTP diphosphokinase gene CRSH1, the carbohydrate-related gene STARCH SYNTHASE 2 (SS2), the cell wall biosynthesis gene CINNAMYL ALCOHOL DEHYDROGENASE (CAD7), and the endocytosis-related gene VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 52 A (VPS52A).

Conclusions: The cold-responsive genes and metabolic pathways of Dendrobium spp. revealed in this study provide important insight to enable the genetic enhancement of cold tolerance in Dendrobium spp., and to facilitate cold tolerance breeding in related plants.

Keywords: Dendrobium spp; Cold stress; Signaling pathway; Transcriptomic patterns; WGCNA.

Fig. 1

Morphological changes of Dendrobium spp plants under 10℃ cold stress. (A) The Dendrobium spp phenotypes. HX, the cold–tolerant genotype ‘Hongxing’; SH, the cold–sensitive genotype “Sonia Hiasakul”; the scale bar is 10 cm. (B) Plant defoliation and (C) Leaf yelllow rate, n = 10. (D) Chlorophyll content and (E) Relative electric conductivity, n = 3. Error bars represent standard deviation. “*” represent a significant differences based on one–way ANOVA followed by Tukey’s multiple comparison (P < 0.05)

Fig. 2

Analysis of physiological indices of HX and SH under 10℃ cold stress. (A) Peroxidase (POD) activity; (B) Malondialdehyde (MDA) content; (C) Free proline (FP) content; (D) Soluble protein (SP) content; (E) Soluble sugar (SS) content. Error bars represent standard deviation (n = 3). “*” represent a significant differences based on one–way ANOVA followed by Tukey’s multiple comparison (P < 0.05)

Fig. 3

The cluster heat map (CHM) (A) and principle component analysis (PCA) (B) of the physiological indices. The numbers 1–3, 4–6, 7–9, 10–12, 13–15 and 16–18 represent three replicates after cold treatment 0, 1, 2, 4, 8 and 16 d, respectively

Fig. 4

The orthogonal partial least squares discriminant analysis (OPLS–DA) of physiological indices. (A) Blue and red lines indicate the distribution of the physiological data in HX and SH, respectively; (B and C) Black dashed lines represent the key nodes of VIP = 1. The numbers 1–3, 4–6, 7–9, 10–12, 13–15 and 16–18 represent three replicates after cold treatment 0, 1, 2, 4, 8 and 16 d, respectively

Fig. 5

Differential and common expression genes in ‘Hongxing’ (HX) and ‘Sonia Hiasakul’ (SH). (A) Volcano plot analysis between two Dendrobium spp cultivars; (B) Analysis of shared genes of HX; (C) Analysis of common genes of SH; (D) Analysis of common genes between HX and SH. HX_1 vs SH_1, HX_2 vs SH_2 and HX_3 vs. SH_3 indicates the comparison between HX and SH at 0, 4 and 8 d, respectively

Fig. 6

Weighted gene co–expression network analysis (WGCNA) of genes related to cold responses in Dendrobium spp. (A) Soft power plot. The abscissa represents the soft threshold (β), the ordinate represents the scale–free topology model fit index (left), and the mean connectivity index (right); (B) Cluster heatmap of all genes in the 11 modules are shown; (C) Clustering dendrogram of genes and module division. The dynamic tree cut represents the module divided according to the expression of each gene, while the merged dynamic is the result of merging similar modules according to the dynamic tree cut; (D) Correlations between modules and traits (time). Each row corresponds to a module, and each module cell contains the corresponding correlation and p-value

Fig. 7

qRT-PCR analysis of 9 cold responsive genes between cold sensitive and cold tolerant genotypes. Error bars represent standard deviation (n = 3). Significant differences between the two cultivars were based on one–way ANOVA followed by Tukey’s multiple comparison (P < 0.05). The Pearson correlation coefficient is expressed as r². CAD7, cinnamyl alcohol dehydrogenase 7; pp34, remorin pp34 protein; PP2C, protein phosphatase 2 C; JAR1, jasmonic acid-amido synthetase 1; CBF1, C-repeat binding factors 1; SS2, starch synthase 2; SKIP17, Ski-interacting protein 17; VPS52A, vacuolar protein sorting-associated protein 52 A; RGA3, disease resistance protein RGA3

Fig. 8

Putative module and cold response pathways in Dendrobium spp. Red boxes indicate cold response factors identified from the co–expression network, while blue boxes represent the validated genes involved in regulating cold tolerance of Dendrobium species

Fig. 9

Analysis of nine hub genes responding to cold stress in Dendrobium spp. Analysis of gene expression patterns responsive to cold stress in Dendrobium spp. PP2C, protein phosphatase 2 C; SnRK2, sucrosenon-fermenting1–relatedproteinkinase 2; ABF1, ABRE binding factors 1; SKIP17, Ski-interacting protein 17; CRSH1, GTP diphosphokinase CRSH1; pp34, remorin pp34 protein; SS2, starch synthase 2; CAD7, Cinnamyl alcohol dehydrogenase 7; VPS52A, vacuolar protein sorting–associated protein 52 A. Red represents upregulated expression genes, and blue indicates downregulated expression genes