In silico analysis identified bZIP transcription factors genes responsive to abiotic stress in Alfalfa (Medicago sativa L.).

BACKGROUND: Alfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies including, but not limited to, differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa. RESULT: In the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in A. thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physico-chemical properties, motif analysis and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. RT-qPCR analysis on selected genes further verified these differential expression patterns. CONCLUSIONS: Taken together, this work provides a framework for the future study of bZIPs in alfalfa and presents candidate bZIPs involved in stress-response signaling.

Starch and sucrose metabolism plays an important role in the stem development in Medicago sativa.

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

Transcriptomic analysis provides insights into the molecular mechanism of melatonin-mediated cadmium tolerance in Medicago sativa L.

Cadmium (Cd), a toxic element, often makes a serious threat to plant growth and development. Previous studies found that melatonin (Mel) reduced Cd accumulation and reestablished the redox balance to alleviate Cd stress in Medicago sativa L., however, the complex molecular mechanisms are still elusive. Here, comparative transcriptome analysis and biochemical experiments were conducted to explore the molecular mechanisms of Mel in enhancing Cd tolerance. Results showed that 7237 differentially expressed genes (DEGs) were regulated by Mel pretreatment to Cd stress compared to the control condition in roots of Medicago sativa L. Besides, in comparison with Cd stress alone, Mel upregulated 1081 DEGs, and downregulated 1085 DEGs. These DEGs were mainly involved in the transcription and translation of genes and folding, sorting and degradation of proteins, carbohydrate metabolism, and hormone signal network. Application of Mel regulated the expression of several genes encoding ribosomal protein and E3 ubiquitin-protein ligase involved in folding, sorting and degradation of proteins. Moreover, transcriptomic analyse suggested that Mel might regulate the expression of genes encoding pectin lyase, UDP-glucose dehydrogenase, sucrose-phosphate synthase, hexokinase-1, and protein phosphorylation in the sugar metabolism. Therefore, these could promote sucrose accumulation and subsequently alleviate the Cd damage. In conclusion, above findings provided the mining of important genes and molecular basis of Mel in mitigating Cd tolerance and genetic cultivation of Medicago sativa L.

The Effect of Genome Parametrization and SNP Marker Subsetting on Genomic Selection in Autotetraploid Alfalfa.

BACKGROUND: Alfalfa, the most economically important forage legume worldwide, features modest genetic progress due to long selection cycles and the extent of the non-additive genetic variance associated with its autotetraploid genome. METHODS: To improve the efficiency of genomic selection in alfalfa, we explored the effects of genome parametrization (as tetraploid and diploid dosages, plus allele ratios) and SNP marker subsetting (all available SNPs, only genic regions, and only non-genic regions) on genomic regressions, together with various levels of filtering on reading depth and missing rates. We used genotyping by sequencing-generated data and focused on traits of different genetic complexity, i.e., dry biomass yield in moisture-favorable (FE) and drought stress (SE) environments, leaf size, and the onset of flowering, which were assessed in 143 genotyped plants from a genetically broad European reference population and their phenotyped half-sib progenies. RESULTS: On average, the allele ratio improved the predictive ability compared with other genome parametrizations (+7.9% vs. tetraploid dosage, +12.6% vs. diploid dosage), while using all the SNPs offered an advantage compared with any specific SNP subsetting (+3.7% vs. genic regions, +7.6% vs. non-genic regions). However, when focusing on specific traits, different combinations of genome parametrization and subsetting achieved better performances. We also released Legpipe2, an SNP calling pipeline tailored for reduced representation (GBS, RAD) in medium-sized genotyping experiments.

Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress.

BACKGROUND: Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown. RESULT: A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1-4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein-protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa. CONCLUSION: This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Mechanism of action of microRNA166 on nitric oxide in alfalfa (Medicago sativa L.) under drought stress.

BACKGROUND: Alfalfa is a perennial forage crop of high importance, but its cultivation is often affected by drought stress. Currently, the investigation of drought-related small RNAs is a popular research topic to uncover plant drought resistance mechanisms. Among these small RNAs, microRNA166 (miR166) is associated with drought in numerous plant species. Initial small RNA sequencing studies have shown that miR166 is highly responsive to exogenous nitric oxide (NO) and drought. Therefore, analyzing the expression of Msa-miR166 under nitric oxide and drought treatment is significant. RESULT: Bioinformatics analysis revealed that the miR166 family is widely distributed among plants, ranging from mosses to eudicots, with significant distribution differences between species. The evolutionary degree of Msa-miR166s is highly similar to that of Barrel medic (Medicago truncatula) and Soybean (Glycine max), but significantly different from the model plant Arabidopsis (Arabidopsis thaliana). It is suggested that there are no significant differences in miR166s within the species, and members of Msa-miR166s can form a typical stem-loop. The lowest level of exogenous nitric oxide was observed in Msa-miR166s under drought stress, followed by individual drought, and the highest level was observed after removing endogenous nitric oxide. CONCLUSION: In response to short-term drought, Msa-miR166s down-regulate expression in alfalfa (Medicago sativa L.). Exogenous nitric oxide can reduce the expression of Msa-miR166s in response to short-term drought. These findings suggest that Msa-miR166e-5p is responsive to environmental changes. The expression levels of target genes showed an opposite trend to Msa-miR166s, verifying the accuracy of Degradome sequencing in the early stage. This suggests that alfalfa experiences drought stress when regulated by exogenous nitric oxide, targeting HD ZIP-III, FRI, and CoA ligase genes. Additionally, the expression of Msa-miR166s in response to drought stress varies between leaves and roots, indicating spatiotemporal specificity.

Regulation of endogenous hormone and miRNA in leaves of alfalfa (Medicago sativa L.) seedlings under drought stress by endogenous nitric oxide.

BACKGROUND: Alfalfa (Medicago sativa. L) is one of the best leguminous herbage in China and even in the world, with high nutritional and ecological value. However, one of the drawbacks of alfalfa is its sensitivity to dry conditions, which is a global agricultural problem. The objective of this study was to investigate the regulatory effects of endogenous nitric oxide (NO) on endogenous hormones and related miRNAs in alfalfa seedling leaves under drought stress. The effects of endogenous NO on endogenous hormones such as ABA, GA3, SA, and IAA in alfalfa leaves under drought stress were studied. In addition, high-throughput sequencing technology was used to identify drought-related miRNAs and endogenous NO-responsive miRNAs in alfalfa seedling leaves under drought stress. RESULT: By measuring the contents of four endogenous hormones in alfalfa leaves, it was found that endogenous NO could regulate plant growth and stress resistance by inducing the metabolism levels of IAA, ABA, GA3, and SA in alfalfa, especially ABA and SA in alfalfa. In addition, small RNA sequencing technology and bioinformatics methods were used to analyze endogenous NO-responsive miRNAs under drought stress. It was found that most miRNAs were enriched in biological pathways and molecular functions related to hormones (ABA, ETH, and JA), phenylpropane metabolism, and plant stress tolerance. CONCLUSION: In this study, the analysis of endogenous hormone signals and miRNAs in alfalfa leaves under PEG and PEG + cPTIO conditions provided an important basis for endogenous NO to improve the drought resistance of alfalfa at the physiological and molecular levels. It has important scientific value and practical significance for endogenous NO to improve plant drought resistance.

Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

Overexpression of the alfalfa (Medicago sativa) gene, MsKMS1, negatively regulates seed germination in transgenic tobacco (Nicotiana tabacum).

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.

Oxalic acid secretion alleviates saline-alkali stress in alfalfa by improving photosynthetic characteristics and antioxidant activity.

Saline-alkali stress significantly affects the growth and yield of alfalfa (Medicago sativa L.). Organic acid secretion is crucial in alleviating abiotic stress-induced damage in plants. In this study, we evaluated the contents of the major organic acids secreted by the roots of tolerant (ZD) and sensitive (LYL) varieties of alfalfa under saline-alkali stress and investigated the effects of these organic acids on the growth, and physiological functions of alfalfa. Our results indicated that the oxalic acid (OA) content was the highest among the organic acids secreted from alfalfa roots under saline-alkali stress, and oxalic acid content was the most significantly different between the two varieties, ZD and LYL, compared to the contents of the other organic acids. Oxalic acid alleviated the inhibition of alfalfa growth caused by saline-alkali stress, improved photosynthetic characteristics, reduced the accumulation of reactive oxygen species, and increased the activity of antioxidant enzymes and content of osmoregulatory substances. Furthermore, oxalic acid resulted in significantly increased expression of genes involved in photosynthesis and antioxidant system in alfalfa under saline-alkali stress. This study revealed the effects of oxalic acid secreted by the root system on stress-related physiological processes, providing valuable insights into the functions of root secretions in plant saline-alkali resistance.

MsSPL12 is a positive regulator in alfalfa (Medicago sativa L.) salt tolerance.

KEY MESSAGE: Over expression of MsSPL12 improved alfalfa salt tolerance by reducing Na+ accumulation and increasing antioxidant enzyme activity and regulating down-stream gene expression. Improvement of salt tolerance is one of the major goals in alfalfa breeding. Here, we demonstrated that MsSPL12, an alfalfa transcription factor gene highly expressed in the stem cells, plays a positive role in alfalfa salt tolerance. MsSPL12 is localized in the nucleus and shows transcriptional activity in the presence of its C-terminus. To investigate MsSPL12 function in plant response to salt stress, we generated transgenic plants overexpressing either MsSPL12 or a chimeric MsSPL12-SRDX gene that represses the function of MsSPL12 by using the Chimeric REpressor gene-Silencing Technology (CRES-T), and observed that overexpression of MsSPL12 increased the salt tolerance of alfalfa transgenic plants associated with an increase in K+/Na+ ratio and relative water content (RWC) under salt stress treatment, but a reduction in electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA), and proline (Pro) compared to wild type (WT) plants. However, transgenic plants overexpressing MsSPL12-SRDX showed an inhibited plant growth and a reduced salt tolerance. RNA-sequencing and quantitative real-time PCR analyses revealed that MsSPL12 affected the expression of plant abiotic resistance-related genes in multiple physiological pathways. The potential MsSPL12-mediated regulatory pathways based on the differentially expressed genes between the MsSPL12 overexpression transgenics and WT controls were predicted. In summary, our study proves that MsSPL12 is a positive regulator in alfalfa salt tolerance and can be used as a new candidate for manipulation to develop forage crops with enhanced salt tolerance.

A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response.

BACKGROUND: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid. RESULTS: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress. CONCLUSIONS: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance.

Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance.

PECTIN ACETYLESTERASE12 regulates shoot branching via acetic acid and auxin accumulation in alfalfa shoots.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

Integrated physiological, metabolomic, and transcriptomic analyses elucidate the regulation mechanisms of lignin synthesis under osmotic stress in alfalfa leaf (Medicago sativa L.).

Alfalfa, an essential forage crop known for its high yield, nutritional value, and strong adaptability, has been widely cultivated worldwide. The yield and quality of alfalfa are frequently jeopardized due to environmental degradation. Lignin, a constituent of the cell wall, enhances plant resistance to abiotic stress, which often causes osmotic stress in plant cells. However, how lignin responds to osmotic stress in leaves remains unclear. This study explored the effects of osmotic stress on lignin accumulation and the contents of intermediate metabolites involved in lignin synthesis in alfalfa leaves. Osmotic stress caused an increase in lignin accumulation and the alteration of core enzyme activities and gene expression in the phenylpropanoid pathway. We identified five hub genes (CSE, CCR, CADa, CADb, and POD) and thirty edge genes (including WRKYs, MYBs, and UBPs) by integrating transcriptome and metabolome analyses. In addition, ABA and ethylene signaling induced by osmotic stress regulated lignin biosynthesis in a contradictory way. These findings contribute to a new theoretical foundation for the breeding of high-quality and resistant alfalfa varieties.

Comparative Analysis of Chloroplast Pan-Genomes and Transcriptomics Reveals Cold Adaptation in Medicago sativa.

Alfalfa (Medicago sativa) is a perennial forage legume that is widely distributed all over the world; therefore, it has an extremely complex genetic background. Though population structure and phylogenetic studies have been conducted on a large group of alfalfa nuclear genomes, information about the chloroplast genomes is still lacking. Chloroplast genomes are generally considered to be conservative and play an important role in population diversity analysis and species adaptation in plants. Here, 231 complete alfalfa chloroplast genomes were successfully assembled from 359 alfalfa resequencing data, on the basis of which the alfalfa chloroplast pan-genome was constructed. We investigated the genetic variations of the alfalfa chloroplast genome through comparative genomic, genetic diversity, phylogenetic, population genetic structure, and haplotype analysis. Meanwhile, the expression of alfalfa chloroplast genes under cold stress was explored through transcriptome analysis. As a result, chloroplast genomes of 231 alfalfa lack an IR region, and the size of the chloroplast genome ranges from 125,192 bp to 126,105 bp. Using population structure, haplotypes, and construction of a phylogenetic tree, it was found that alfalfa populations could be divided into four groups, and multiple highly variable regions were found in the alfalfa chloroplast genome. Transcriptome analysis showed that tRNA genes were significantly up-regulated in the cold-sensitive varieties, while rps7, rpl32, and ndhB were down-regulated, and the editing efficiency of ycf1, ycf2, and ndhF was decreased in the cold-tolerant varieties, which may be due to the fact that chloroplasts store nutrients through photosynthesis to resist cold. The huge number of genetic variants in this study provide powerful resources for molecular markers.