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Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Understanding the mechanisms underlying the responses to inorganic phosphate (Pi) deficiency in alfalfa will help enhance Pi acquisition efficiency and the sustainable use of phosphorous resources. Integrated global metabolomic and transcriptomic analyses of mid-vegetative alfalfa seedlings under 12-day Pi deficiency were conducted. Limited seedling growth were found, including 13.24%, 16.85% and 33.36% decreases in height, root length and photosynthesis, and a 24.10% increase in root-to-shoot ratio on day 12. A total of 322 and 448 differentially abundant metabolites and 1199 and 1061 differentially expressed genes were identified in roots and shoots. Increased (>3.68-fold) inorganic phosphate transporter 1;4 and SPX proteins levels in the roots (>2.15-fold) and shoots (>2.50-fold) were related to Pi absorption and translocation. The levels of phospholipids and Pi-binding carbohydrates and nucleosides were decreased, while those of phosphatases and pyrophosphatases in whole seedlings were induced under reduced Pi. In addition, nitrogen assimilation was affected by inhibiting high-affinity nitrate transporters (NRT2.1 and NRT3.1), and nitrate reductase. Increased delphinidin-3-glucoside might contribute to the gray-green leaves induced by Pi limitation. Stress-induced MYB, WRKY and ERF transcription factors were identified. The responses of alfalfa to Pi deficiency were summarized as local systemic signaling pathways, including root growth, stress-related responses consisting of enzymatic and nonenzymatic systems, and hormone signaling and systemic signaling pathways including Pi recycling and Pi sensing in the whole plant, as well as Pi recovery, and nitrate and metal absorption in the roots. This study provides important information on the molecular mechanism of the response to Pi deficiency in alfalfa., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Background: Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa's productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress., Results: In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24 h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551 bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress., Conclusions: Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

Alfalfa is the most extensively cultivated forage legume worldwide. However, the molecular mechanisms underlying alfalfa responses to exogenous abscisic acid (ABA) are still unknown. In this study, the first global transcriptome profiles of alfalfa roots under ABA treatments for 1, 3 and 12 h (three biological replicates for each time point, including the control group) were constructed using a BGISEQ-500 sequencing platform. A total of 50,742 isoforms with a mean length of 2541 bp were generated, and 4944 differentially expressed isoforms (DEIs) were identified after ABA deposition. Metabolic analyses revealed that these DEIs were involved in plant hormone signal transduction, transcriptional regulation, antioxidative defense and pathogen immunity. Notably, several well characterized hormone signaling pathways, for example, the core ABA signaling pathway, was activated, while salicylic acid, jasmonate and ethylene signaling pathways were mainly suppressed by exogenous ABA. Moreover, the physiological work showed that catalase and peroxidase activity and glutathione and proline content were increased after ABA deposition, which is in accordance with the dynamic transcript profiles of the relevant genes in antioxidative defense system. These results indicate that ABA has the potential to improve abiotic stress tolerance, but that it may negatively regulate pathogen resistance in alfalfa.

S-adenosylmethionine synthetase (SAMS) is the key enzyme catalysing the formation of S-adenosylmethionine (SAM), a precursor of polyamines and ethylene. To investigate the potential role of SAMS in cold tolerance, we isolated MfSAMS1 from the cold-tolerant germplasm Medicago sativa subsp. falcata and analysed the association of SAM-derived polyamines with cold tolerance. The expression of MfSAMS1 in leaves was greatly induced by cold, abscisic acid (ABA), H2O2 and nitric oxide (NO). Our data revealed that ABA, H2O2 and NO interactions mediated the cold-induced MfSAMS1 expression and cold acclimation in falcata. SAM, putrescine, spermidine and spermine levels, ethylene production and polyamine oxidation were sequentially altered in response to cold, indicating that SAMS-derived SAM is preferentially used in polyamine synthesis and homeostasis during cold acclimation. Antioxidant enzyme activities were also induced in response to cold and showed correlation with polyamine oxidation. Overexpression of MfSAMS1 in tobacco resulted in elevated SAM levels, but polyamine levels and ethylene production in the transgenic plants were not significantly changed. Compared to the wild type, transgenic plants had increased levels of apoplastic H2O2, higher transcript levels of genes involved in polyamine synthesis and oxidation, and higher activities of polyamine oxidation and antioxidant enzymes. The results showed that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2 O2 -induced antioxidant protection, as a result enhanced tolerance to freezing and chilling stress in transgenic plants. This is the first report demonstrating that SAMS plays an important role in plant tolerance to cold via up-regulating polyamine oxidation., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar 'XinjiangDaye' using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources. [ABSTRACT FROM AUTHOR]

Aquaporins (AQP) are distributed ubiquitously in plants, and they play important roles in multiple aspects of plant growth and development, as well as in plant resistance to various environmental stresses. In this study, 43 MsAQP genes were identified in the forage crop Medicago sativa. All the MsAQP proteins were clustered into four subfamilies based on sequence similarity and phylogenetic relationship, including 17 TIPs, 14 NIPs, 9 PIPs and 3 SIPs. Analyses of gene structure and conserved domains indicated that the majority of the deduced MsAQP proteins contained the signature transmembrane domains and the NPA motifs. Analyses on cis-acting elements in the promoter region of MsAQP genes revealed the presence of multiple and diverse stress-responsive and hormone-responsive cis-acting elements. In addition, by analyzing the available and comprehensive gene expression data of M. truncatula, we screened ten representative MtAQP genes that were responsive to NaCl or drought stress. By analyzing the sequence similarity and phylogenetic relationship, we finally identified the corresponding ten salt- or drought-responsive AQP genes in M. sativa, including three MsTIPs, three MsPIPs and four MsNIPs. The qPCRs showed that the relative expression levels of these ten selected MsAQP genes responded differently to NaCl or drought treatment in M. sativa. Gene expression patterns showed that most MsAQP genes were preferentially expressed in roots or in leaves, which may reflect their tissue-specific functions associated with development. Our results lay an important foundation for the future characterization of the functions of MsAQP genes, and provide candidate genes for stress resistance improvement through genetic breeding in M. sativa. [ABSTRACT FROM AUTHOR]

Phosphite, a reduced form of phosphate, inhibits the growth and even has toxic effect on plants. To learn more about the mechanism of alfalfa responses to phosphite, the morphological and physiological characteristics, and the metabolites and transcript levels were comprehensively analyzed following the exposure of alfalfa seedlings to phosphite and phosphate under greenhouse conditions. The results showed that phosphite inhibited seedling growth and photosynthesis. However, the absorption efficiency of phosphite was higher than that of phosphate in roots, which was supported by increased total phosphorus concentration of 16.29% and 52.30% on days 8 and 12. Moreover, phosphite stress affected the synthesis of lipids and carbohydrates, which were reflected in enhanced glycolipid and sulfolipid in roots and amylose in shoots. Phosphite stress resulted in a decrease in indole acetic acid (IAA) in the whole plant and zeatin in the shoots, which could enable alfalfa to adapt to the phosphite environment. Some genes involved in phosphate starvation response included SPX , phosphate response regulator2 , and inorganic phosphate transporter 1–4 (PHT1;4) in roots were affected by phosphite stress. In addition, some genes that are involved in stress responses and DNA repair were induced by phosphite stress. These observations together suggest that alfalfa responds to phosphite stress by inhibiting growth, regulating the genes induced by phosphate starvation, improving oxidative protection, promoting DNA repair, and adjusting the IAA and zeatin signaling transductions. Our findings provide novel insights into the molecular response to phosphite stress in alfalfa. • Phosphite inhibited the growth of alfalfa seedlings. • Phosphite inhibited nitrogen assimilation and promoted sulfate assimilation. • Phosphite stress increased Phosphate starvation induced (PSI) genes. • Phosphite induced stress response genes and promoted DNA repair in alfalfa. [ABSTRACT FROM AUTHOR]