Study on molecular response of alfalfa to low temperature stress based on transcriptomic analysis

Abstract

Background: Alfalfa (Medicago sativa L.) is an important high-quality forage crop. Low temperature is an abiotic stress factor that affects the distribution and productivity of alfalfa. To further understand the molecular response to low temperature, and to identify additional genes and metabolic pathways associated with cold tolerance in alfalfa, in this study we conducted transcriptome sequencing, weighted gene co-expression network analysis, KEGG pathway enrichment analysis, and quantitative real-time PCR validation in alfalfa cultivars subjected to low-temperature treatment.

Results: Weighted gene co-expression network analysis revealed that three gene modules were significantly negatively correlated with the semi-lethal temperature for alfalfa. Genes in the three modules were used to construct gene co-expression networks, from which MS.gene46105, MS.gene044087, MS.gene76894, MS.gene44620, MS.gene22005, MS.gene045060, MS.gene31405, and MS.gene74761 were selected as important genes associated with cold tolerance. Quantitative real-time PCR analysis of these eight genes validated the reliability of the transcriptome sequencing data. In addition, further analysis of the genes within the three modules revealed that several transcription factors (AP2/ERF, bZIP, C3H, NAC, and others) and metabolic pathways (N-glycan biosynthesis, citrate cycle, glycolysis/gluconeogenesis, and carbon metabolism, and others) responded well to the low temperature.

Conclusions: Three gene modules, eight genes, several transcription factors and multiple metabolic pathways associated with cold tolerance were screened. This results will provide a valuable reference for further clarification of the cold tolerance mechanism and breeding for cold tolerance in alfalfa.

Keywords: Alfalfa; Cold tolerance; Low temperature; Molecular response; Weighted gene co-expression network analysis.

Fig. 1

Semi-lethal temperature (LT₅₀) of alfalfa crowns. A smaller LT₅₀ value indicates stronger cold tolerance. Different letters indicate a significant difference in LT₅₀ between different sampling time points

Fig. 2

Results of weighted gene co-expression network analysis. (A) Selection of the soft threshold. The left panel depicts the log k value on the vertical axis, representing the square of the correlation coefficient, and the horizontal axis is the weight parameter β (soft threshold). The right panel shows the mean connectivity of all genes in the corresponding gene module on the vertical axis. (B) Hierarchical clustering dendrogram, where each color in the tree represents a module, and each gene belonging to the same module is indicated by the corresponding color. The vertical distance is the distance between two genes, and the horizontal distance is not meaningful. (C) Correlations between gene modules and the cold tolerance of alfalfa. Darker colors indicate stronger correlations. The numbers outside the parentheses are the correlation coefficients, and the numbers inside the parentheses represent the significance of the correlations

Fig. 3

Classification of related transcription factors in three gene modules. The numbers outside parentheses are the number of related genes involved. The numbers in parentheses are the proportion of the number of genes (195) in this statistic

Fig. 4

Scatterplots of KEGG pathway enrichment analysis of genes within three cold tolerance-associated gene modules. (A) Brown, (B) darkgreen, and (C) red gene modules. The vertical axis represents KEGG pathways, and the horizontal axis is the ratio of ‘the number of differentially expressed genes annotated with the pathway term’ to ‘the total number of genes annotated with the pathway term’ (rich factor). A larger rich factor indicates a higher degree of enrichment. The size of the points corresponds to the number of genes enriched in the pathway. The color of the points represents the significance of pathway enrichment, assessed using the q-value. A q-value closer to 0 is indicated by a redder color, representing a more significant enrichment. The figure displays the top 20 significantly enriched pathways

Fig. 5

Gene co-expression networks of partial genes within the (A) red, (B) darkgreen, and (C) brown gene modules. The red nodes represent the hub genes, and the yellow nodes represent other genes in the gene networks with relatively high connection strength

Fig. 6

FPKM values derived from transcriptome sequencing data for eight cold tolerance-associated genes at six time points during cold treatment. The x-axis is the sampling time points and the y-axis is the gene expression level. Different letters indicate a significant difference between time points within the same cultivar (P < 0.05)

Fig. 7

Expression levels of eight selected cold tolerance-associated genes during cold treatment in the alfalfa cultivars ‘WL168HQ’, ‘WL343HQ’, and ‘WL440HQ’. The x-axis is the sampling time points and the y-axis is the gene expression level. Different letters indicate a significant difference in gene expression level between different time points within the same cultivar (P < 0.05)