Alfalfa (Medicago sativa) is the most widely grown and most important forage crop in the world. However, alfalfa is susceptible to waterlogging stress, which is the major constraint for its cultivation area and crop production. So far, the molecular mechanism of alfalfa response to the waterlogging is largely unknown. Here, comparative transcriptome combined with proteomic analyses of two cultivars (M12, tolerant; M25, sensitive) of alfalfa showing contrasting tolerance to waterlogging were performed to understand the mechanism of alfalfa in response to waterlogging stress. Totally, 748 (581 up- and 167 down-regulated) genes were differentially expressed in leaves of waterlogging-stressed alfalfa compared with the control (M12_W vs. M12_CK), whereas 1193 (740 up- and 453 down-regulated) differentially abundant transcripts (DATs) were detected in the leaves of waterlogging-stressed plants in comparison with the control plants (M25_W vs. M25_CK). Furthermore, a total of 187 (122 up- and 65 down-regulated) and 190 (105 up- and 85 down-regulated) differentially abundant proteins (DAPs) were identified via isobaric tags for relative and absolute quantification (iTRAQ) method in M12_W vs. M12_CK and M25_W vs. M25_CK comparison, respectively. Compared dataset analysis of proteomics and transcriptomics revealed that 27 and eight genes displayed jointly up-regulated or down-regulated expression profiles at both mRNA and protein levels in M12_W vs. M12_CK comparison, whereas 30 and 27 genes were found to be co-up-regulated or co-down-regulated in M25_W vs. M25_CK comparison, respectively. The strongly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for co-up-regulated genes at mRNA and protein levels in M12_W vs. M12_CK comparison were ‘Amino sugar and nucleotide sugar metabolism’, ‘Arginine and proline metabolism’ and ‘Starch and sucrose metabolism’, whereas co-up-regulated protein-related pathways including ‘Arginine and proline metabolism’ and ‘Valine, leucine and isoleucine degradation’ were largely enriched in M25_W vs. M25_CK comparison. Importantly, the identified genes related to beta-amylase, Ethylene response Factor (ERF), Calcineurin B-like (CBL) interacting protein kinases (CIPKs), Glutathione peroxidase (GPX), and Glutathione-S-transferase (GST) may play key roles in conferring alfalfa tolerance to waterlogging stress. The present study may contribute to our understanding the molecular mechanism underlying the responses of alfalfa to waterlogging stress, and also provide important clues for further study and in-depth characterization of waterlogging-resistance breeding candidate genes in alfalfa.

The objective was to determine effects of cellulase, xylanase, and commercial fibrolytic enzymes on fermentation quality, aerobic stability, bacterial community, and in vitro degradation of mixed silages. Mixtures of alfalfa, wheat bran, and rice straw [80:15:5 on a fresh matter (FM) basis] were ensiled for 1, 3, 5, 7, 15, 30, and 45 d after treatment with: distilled water (control, C); cellulase (E); xylanase (X); or commercial fibrolytic enzymes (EX), with all enzyme preparations applied at 100 U/g FM. The 45-day silages were subjected to an in vitro degradation test. Each of the three enzyme-treated groups enriched relative abundance (RA) of Lactobacillus, Weissella, and Stenotrophomonas maltophilia, increased water soluble carbohydrate (WSC) concentrations, and extended aerobic stability over 384 h, but concurrently inhibited growth of undesirable microbes (i.e., Acinetobacter sp, Lelliottia amnigena, and Sphingomonas sp), reducing pH and concentrations of ammonia nitrogen (AN), butyric acid (BA) and propionic acid (PA). Compared to C, adding X or EX increased the RA of L. paralimentarius and L. parabrevis, enhanced accumulation of acetic acid (AA) and crude protein (CP), and reduced hemicellulose content. Furthermore, E group silage had the highest abundance of W. cibaria. In addition, EX enriched RA of Bacillus velezensis, reduced AN concentration, increased DM degradability, total VFA production, and gas production during in vitro incubation. In conclusion, addition of X or EX enhanced ensiling by enhancing concentrations of AA; however, EX was the most promising enzyme, based on reducing AN concentration and increasing DM content and DM degradability. Graphical Abstract

In nature, some plant species produce seedpods with spines, which is an adaptive biological trait for protecting the seed and helping seed dispersal. However, the molecular mechanism of spine formation is still unclear. While conducting routine tissue culture and transformation in the model legume Medicago truncatula, we identified a smooth seedpod (ssp1) mutant with a suite of other phenotypic changes. Preliminary analysis showed that the mutation was derived from the tissue culture process. Genetic segregation analysis suggested that ssp1 is a recessive mutant. By combining whole-genome sequencing and bioinformatics analysis, we found that the mutant phenotype was caused by a single nucleotide polymorphism and a 30 bp deletion in the gene locus Medtr4g039430, named SSP1. Complementation of the M. truncatula ssp1 and Arabidopsis twd1 mutants showed complete restoration, indicating that SSP1 is an ortholog of Arabidopsis TWD1 which encodes an immunophilin-like FK506-binding protein 42. The formation of spines on seedpods is associated with auxin transport. The method used in this study offers an effective way for detecting genes responsible for somaclonal variations. The results demonstrate, for the first time, that SSP1 plays a crucial role in the determination of spine formation on seedpods.