Comparative transcriptome analysis of differentially expressed genes of Medicago falcata L. breeding lines response to saline-alkaline stress.

Background: Salt-alkali stress is an abiotic stress that inhibits crop growth and reduces yield. It significantly affects various physiological processes in plants, including photosynthesis, osmotic regulation, and antioxidant defense. However, studies on the transcriptional response mechanisms of Medicago falcata L. under salt-alkali stress are limited. In this study, RNA-seq technology was used to analyze differentially expressed genes (DEGs) in salt-alkali tolerant M.falcata breeding lines (LM18) and the salt-alkali sensitive Hulunbeier (HL) under salt-alkali stress. Furthermore, physiological indicators such as chlorophyll content, proline accumulation, and superoxide dismutase (SOD) activity were assessed to compare the responses of LM18 and HL to salt-alkali stress. By integrating transcriptomic and physiological analyses, this study provides new insights into the physiological and molecular regulatory mechanisms of M. falcata in response to salt-alkali stress.

Results: The results showed that compared to the untreated controls, 10,289 and 2,478 DEGs were detected in LM18 and HL M.falcata seedlings, with 788 shared DEGs detected in both. GO functional analysis classified these DEGs into three categories: Biological Process, Cellular Components, and Molecular Functions, with significant enrichment in GO terms such as "response to osmotic stress", "intramolecular oxidoreductase activity" and "antioxidant activity". Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the involvement of these DEGs in key metabolic pathways, including "Phenylpropanoid biosynthesis", "Plant hormone signal transduction", "Plant-pathogen interaction", "Isoflavonoid biosynthesis", "Circadian rhythm-plant" and "Photosynthesis-antenna proteins". Physiological indicators and membership function analysis confirmed that LM18 has greater salt-alkali tolerance than HL. Transcription factor analysis identified 42 transcription factor families, with the ERF family being the most abundant, followed by MYB-related, WRKY, bHLH, and MYB families. Weighted Gene Co-expression Network Analysis (WGCNA) showed that the MEturquoise module exhibited a significant positive correlation with salt-alkali stress and several physiological indicators. Module gene network analysis and GO enrichment revealed that MS.gene64536(MYBP), MS.gene76249(SRM1) and MS.gene049843 (MPK3) have functions related to "response to salt stress" and "positive regulation of response to salt stress", suggesting their key roles in salt-alkali tolerance in M.falcata. All three genes were upregulated in the salt-alkali tolerant LM18.

Conclusions: The GO terms and KEGG pathways significantly enriched in LM18 involved a significantly higher number of DEGs compared to HL, suggesting a more robust and effective mechanism in LM18. These findings highlight the robust molecular and physiological adaptations of LM18 in response to salt-alkali stress.