De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress

Abstract

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen species and malondialdehyde and a decreased content of chlorophyll, indicating that anti-oxidation and detoxification play an important role in response to saline-alkaline stress. Overall, the transcriptome analysis provided novel insights into the saline-alkaline stress tolerance response mechanisms in alfalfa.

Keywords: Medicago sativa; flavonol; ion torrent sequencing; peroxidase; saline-alkaline stress; transcription factors.

FIGURE 1

Venn diagram of the number of differentially expressed genes (DEGs) (log₂ > 1.5) in different time points in de novo transcriptome (false discovery rate < 0.025). (A) Distribution of all DEG (B) up-regulated DEG; and (C) down-regulated DEG annotated in samples treated with alkaline-solution for 1 day (red circles) and 7 days (blue circles).

FIGURE 2

Validation of next generation sequencing (NGS data by quantitative reverse transcriptase PCR (qRT-PCR) in samples treated with alkaline-saline solution for 1 day and 7 days.

FIGURE 3

Histogram of gene ontology (GO) categories in response to 1 day and 7 days of treatment with saline-alkaline stress. The bars represent log₂-transformed ratio between category portions in up-regulated and down-regulated gene sets.

FIGURE 4

Hierarchical cluster analysis of differentially expressed genes based on expression pattern of 53,853 genes derived from the control, 1 day and 7 days treatments.

FIGURE 5

Transcription factors differentially expressed in samples after 1 day and 7 days of saline-alkaline stress.

FIGURE 6

Expression pattern of genes in response to saline-alkaline stress and saline stress after 7 days of treatment. a, inositol transporter; b, DNA binding protein; c, raffinose synthase; d, ferritin; e, ACC synthetase; f, NAC transcription factor; g, glutathione S-transferase; h, xyloglucan endotransglucosylase.

FIGURE 7

Physiology assay of alfalfa under saline-alkaline stress. (A) content of ${\text{O}}_2^{-}$; (B) content of H₂O₂; (C) content of MDA; (D) activity of POD; (E) content of chlorophyll.

FIGURE 8

Activity of AKR in root part under saline stress and saline-alkaline stress respectively.