Deciphering the molecular signatures of tropical Areca catechu L. under cold stress: an integrated physiological and transcriptomic analysis

Abstract

Introduction: Areca catechu is a widely cultivated palm species with significant economic and medicinal value. However, A. catechu is a tropical plant that is particularly susceptible to low temperatures.

Methods: This study integrates physiological profiling with transcriptomic sequencing to systematically investigate the cold-response mechanisms of A. catechu.

Results: Multivariate variance analysis revealed that peroxidase (POD) activity and chlorophyll content are significant biomarkers strongly correlated with cold tolerance. A comprehensive investigation into the temporal expression of genes in response to 24 hours of cold stress was conducted, using RNA-seq analysis. This analysis yielded a substantial number of differentially expressed genes (DEGs), amounting to 20,870, which were found to be subject to temporal regulation. KEGG pathway enrichment analysis revealed substantial activation in three metabolic pathways: phytohormone signaling, alkaloid biosynthesis (tropane/piperidine/pyridine), and flavonoid biosynthesis. The application of Weighted Gene Co-expression Network Analysis (WGCNA), in conjunction with a dynamic tree-cutting algorithm, resulted in the identification of 25 co-expression modules. Eigenvector centrality analysis identified six hub genes responsive to cold stress: ZMYND15, ABHD17B, ATL8, WNK5, XTH3 and TPS. The findings of this study delineate three key aspects: (1) temporal dynamics of cold-responsive physiological processes, (2) pathway-level characterization of DEG enrichment patterns, and (3) genetic determinants underlying cold stress adaptation.

Discussion: These findings clarify the time series and core physiological indicators of A. catechu during various physiological processes, identify pivotal genes associated with cold stress, and provide a gene-to-phenotype framework for optimizing cold-resilient cultivation protocols and molecular marker-assisted breeding strategies.

Keywords: Areca catechu L.; RNA-seq; WGCNA; cold stress; multivariate analysis.

Figure 1

Physiological response of A catechu to cold stress. (A) Peroxidase (POD). (B) superoxide dismutase (SOD). (C) Malondialdehyde (MDA). (D) Catalase (CAT). (E) Hydrogen peroxide (H₂O₂). (F) Free Proline. (G) Soluble sugar (SS). (H) Soluble protein (SP). C0d denotes A. catechu grown at 26°C (control), where Figures (A-H) Data were analyzed using Tukey’s method of multiple comparisons test at a significance level of P < 0.05. Lowercase letters (a, b, c) indicate statistically homogeneous groups based on Tukey’s multiple comparison test (P<0.05). Groups sharing the same letter are not significantly different, while those with different letters show significant differences.

Figure 2

Phenotypic changes in leaves of A. catechu after low temperature stress and analysis of data related to phenotype. (A) Phenotypic changes in the leaves of A. catechu at 6 days and 10 days after cold stress. (B) Changes in chlorophyll content of A. catechu leaves. (C) Changes in the net photosynthetic rate of A. catechu. (D) Changes in leaf relative conductivity (E) Changes in relative leaf water content. C0d (0 days) indicates A. catechu grown at 26°C (control). Data were analyzed using Tukey’s method of multiple comparison test at a significance level of P < 0.05. Lowercase letters (a, b, c) indicate statistically homogeneous groups based on Tukey’s multiple comparison test (P<0.05). Groups sharing the same letter are not significantly different, while those with different letters show significant differences.

Figure 3

Multivariate analysis of physiological indices of A. catechu. (A) Clustering heat map of physiological indicators of A. catechu. (B) Principal component analysis (PCA) of physiological indices of A. catechu. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) scatterplots. (D) Orthogonal partial least squares discriminant analysis (OPLS-DA) VIP scores.

Figure 4

Identification and GO enrichment analysis of differentially expressed genes in A. catechu after cold stress. (A) Inter-sample principal component analysis. (B) DEGs for two-by-two comparisons between samples at different time points. (C) Venn diagram of group I DEGs. (D) Venn diagram of group II DEGs. (E) GO enrichment analysis of DEGs in group I (F) GO enrichment analysis of DEGs in group II.

Figure 5

KEGG pathway enrichment analysis of DEGs under cold stress. (A) Group II DEGs; (B) Group II DEGs. Column color gradient indicates the significance of enrichment, where P< 0.001 is marked as ***, P< 0.01 is marked as **, and P< 0.05 is marked as *.

Figure 6

WGCNA of cold stress-related genes in A catechu. (A) Module division of gene expression trends, where a dendrite represents a gene and a color represents a module. (B) Correlation between modules and samples. The horizontal coordinates represent different samples, and the vertical coordinates represent different modules. (C-H) Six different modules, each containing one core gene, were screened in the co-expression network. These core genes may be core genes associated with cold stress in A catechu. (A) TRINITY_DN30068_c0_g1 (ZMYND15) (B) TRINITY_DN979_c0_g2 (ABHD17B) (C) TRINITY_DN21301_c0_g2 (ATL8) (D) TRINITY_DN42763_c0_g1 (WNK5) (E) TRINITY_DN9633_c0_g1 (XTH3) (F) TRINITY_DN6894_c1_g1 (TPS).

Figure 7

RT-qPCR verified the expression pattern of key hub genes. Data were analyzed using Tukey’s multiple comparison test at a significance level of P < 0.05, and error lines indicate standard errors. (A) ZMYND15: MYND-containing zinc binding protein 15. (B) ABHD17B: Hydrolysis enzyme-containing structural domain protein 17B. (C) ATL8: E3 ubiquitin ligase. (D) WNK5: Serine/Threonine Protein Kinase. (F) XTH3: xyloglucan endotransglucosylase/hydrolase 3 (E) TPS, trehalose-phosphate synthase. Lowercase letters (a, b, c) indicate statistically homogeneous groups based on Tukey’s multiple comparison test (P<0.05). Groups sharing the same letter are not significantly different, while those with different letters show significant differences.

Figure 8

Line plots of the parameters of interest (POD, SOD, CAT, and Chlorophyll) as a function of stress time are presented, with the slope K representing the gradient between 0-2days, 2-6days, and 6-10days values.

Figure 9

Gene expression profiles related to key synthetic pathways of cold stress in A catechu. The numbers in the legend indicate the log₂FC value of the gene after treatment compared to the control (C0h). (A) Expression profiles of key genes in the growth hormone signaling pathway. (B) Expression profiles of key genes of the alkaloid synthesis pathway, GOT, ASP, LS, and PPAR. (C) Expression profiles of key genes in the flavonoid biosynthetic pathway. (D) Expression profiles of key genes in the alginate synthesis pathway. (A) AUX1, Auxin-Resistant 1; TIR1: Transport inhibitor response protein 1; AUX/IAA, growth hormone primary response genes; ARF, Auxin Response Factors; GH3, growth hormone amide synthase gene; SAUR, growth hormone up-regulated small RNA gene. (B) ASP, Asparagine synthetase; GOT, Aspartate aminotransferase; PPAR, Peroxisome Proliferator-Activated Receptor; LS, Littorina Synthase; CYP, enzyme tropine synthase; H6H, ranolazine 6B-hydroxylase. (C) PAL, phenylalanine deaminase; CHS, chalcone synthase; CHl, chalcone isomerase; F3H, flavonol 3-hydroxylase; F3’H, flavonol 3’-hydroxylase; DFR, dihydroflavonol 4-reductase; ANR, erythrocyanidin reductase; ANS, anthocyanin synthase; 3GT, flavonol 3-0-glucosyltransferase; 3RT, cornflavonoid 3-rutinoside. (D) TPS, trehalose phosphate synthase; T6P, trehalose-6-phosphate.

Figure 10

Proposed cold adaptation network for A. catechu. Dashed lines indicate inferred interactions.