Fengling Shi

BACKGROUND: Medicago ruthenica, a wild and perennial legume forage widely distributed in semi-arid grasslands, is distinguished by its outstanding tolerance to environmental stress. It is a close relative of commonly cultivated forage of alfalfa (Medicago sativa). The high tolerance of M. ruthenica to environmental stress makes this species a valuable genetic resource for understanding and improving traits associated with tolerance to harsh environments. RESULTS: We sequenced and assembled genome of M. ruthenica using an integrated approach, including PacBio, Illumina, 10×Genomics, and Hi-C. The assembled genome was 904.13 Mb with scaffold N50 of 99.39 Mb, and 50,162 protein-coding genes were annotated. Comparative genomics and transcriptomic analyses were used to elucidate mechanisms underlying its tolerance to environmental stress. The expanded FHY3/FAR1 family was identified to be involved in tolerance of M. ruthenica to drought stress. Many genes involved in tolerance to abiotic stress were retained in M. ruthenica compared to other cultivated Medicago species. Hundreds of candidate genes associated with drought tolerance were identified by analyzing variations in single nucleotide polymorphism using accessions of M. ruthenica with varying tolerance to drought. Transcriptomic data demonstrated the involvements of genes related to transcriptional regulation, stress response, and metabolic regulation in tolerance of M. ruthenica. CONCLUSIONS: We present a high-quality genome assembly and identification of drought-related genes in the wild species of M. ruthenica, providing a valuable resource for genomic studies on perennial legume forages.

Drought is a major abiotic stress that threatens crop production. Soil microbiomes are thought to play a role in enhancing plant adaptation to various stresses. However, it remains unclear whether soil microbiomes play a key role when plants are challenged by drought and whether different varieties are enriched with specific bacteria at the rhizosphere. In this study, we measured changes in growth phenotypes, physiological and biochemical characteristics of drought-tolerant alfalfa (AH) and drought-sensitive (QS) under sterilized and unsterilized soil conditions with adequate watering and with drought stress, and analyzed the rhizosphere bacterial community composition and changes using 16S rRNA high-throughput sequencing. We observed that the unsterilized treatment significantly improved the growth, and physiological and biochemical characteristics of alfalfa seedlings under drought stress compared to the sterilized treatment. Under drought stress, the fresh and dry weight of seedlings increased by 35.24, 29.04, and 11.64%, 2.74% for unsterilized AH and QS, respectively, compared to sterilized treatments. The improvement was greater for AH than for QS. AH and QS recruited different rhizosphere bacteria when challenged by drought. Interestingly, under well-watered conditions, the AH rhizosphere was already rich in drought-tolerant bacterial communities, mainly Proteobacteria and Bacteroidetes , whereas these bacteria started to increase only when QS was subjected to drought. When drought stress was applied, AH was enriched with more drought-tolerant bacteria, mainly Acidobacteria , while the enrichment was weaker in QS rhizosphere. Therefore, the increase in drought tolerance of the drought-tolerant variety AH was greater than that of the drought-sensitive variety QS. Overall, this study confirmed the key role of drought-induced rhizosphere bacteria in improving the adaptation of alfalfa to drought stress, and clarified that this process is significantly related to the variety (genotype). The results of this study provide a basis for improving drought tolerance in alfalfa by regulating the rhizosphere microbiome.

Flavonoids are the major secondary metabolites participating in many biological processes of plants. Although flavonoid biosynthesis has been extensively studied, its regulatory mechanisms during the day and night cycles remain poorly understood. In this study, three proteins, MsMYB206, MsMYB450, and MsHY5, were found to interact with each other, in which MsMYB206 directly transactivated two flavonoid biosynthetic genes, MsFLS and MsF3'H. The expression patterns of MsMYB206, MsMYB450, MsFLS, and MsF3'H were fully consistent at regular intervals across day/night cycles that were higher at night than in the daytime. On the contrary, both gene expression levels and protein contents of MsHY5 increased in the daytime but decreased at night, and the lower expression of MsHY5 at night led to strengthened interaction between MsMYB206 and MsMYB450. The MsMYB206-overexpression plants were more salt-tolerant and their flavonoid contents were higher than the WT during the day/night cycles. This study revealed one mechanism interpreting the fluctuating flavonoid contents during day/night cycles regulated by the MsMYB206/MsMYB450/MsHY5-MsFLS/MsF3'H module that also contributed to salt tolerance in alfalfa.