Overexpression of P5CDH from Cleistogenes songorica improves alfalfa growth performance under field drought conditions

Water stress affects the metabolic regulation and delays the growth and development of alfalfa, causing a reduction in biomass. New alfalfa germplasm was created with improved drought tolerance in greenhouse conditions by introducing the key gene P5CDH1 from C. songorica, a xerophytic grass. However, the field adaptability and response mechanism of new drought-tolerant alfalfa germplasms under water stress are still unclear. In the present study, the yield and quality traits of transgenic CsP5CDH1 alfalfa lines under water stress and normal irrigation conditions were measured and analyzed for two years. The genetic variance components of the tested traits were calculated from the data fitted by the mixed linear model. The plant height of all lines showed significant genotypic variation (σ2g) (P < 0.05), and the stem diameter, stem number, and dry weight of all lines had a significant genotype × environment interaction (σ2ge) (P < 0.05). The heritability (H) of plant height, stem diameter, stem number, dry weight and leaf-to-stem ratio of alfalfa lines were 0.87, 0.52, 0.59, 0.52 and 0.50, respectively. There were significant genotype × environment interactions (σ2ge) (P < 0.05) for the quality traits of all lines. The heritabilities (H) of acid detergent fiber and neutral detergent fiber were 0.65 and 0.64, respectively. The results of transcriptional expression analysis with RNA-seq showed that the genes MsProDH1, MsProDH4, MsProDH5, MsP5CDH1, MsP5CS5, MsP5CS9, and MsP5CR1, which are involved in the proline metabolism pathway, played an important role in the drought tolerance of innovative alfalfa germplasm. Under water stress, with the regulation of key genes in the proline metabolism pathway, the proline content of all alfalfa lines increased to varying degrees. Among them, the proline content in the shoots and roots of transgenic line L6 was 7.29 times and 12.22 times that under normal irrigation conditions, respectively. The present study helped to clarify that the new germplasm of alfalfa transformed with the CsP5CDH gene synthesized a large amount of proline under water stress, and effectively slowed leaf water loss, thus improving the drought resistance of alfalfa.