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.

A genome-wide association study reveals novel loci and candidate genes associated with plant height variation in Medicago sativa.

BACKGROUND: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide. RESULTS: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6__31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development. CONCLUSIONS: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa.

Overexpression of the elongation factor MtEF1A1 promotes salt stress tolerance in Arabidopsis thaliana and Medicago truncatula.

BACKGROUND: Elongation factor 1 A (EF1A), an essential regulator for protein synthesis, has been reported to participate in abiotic stress responses and environmental adaption in plants. However, the role of EF1A in abiotic stress response was barely studied in Medicago truncatula. Here, we identified elongation factor (EF) genes of M. truncatula and studied the salt stress response function of MtEF1A1 (MTR_6g021805). RESULTS: A total of 34 EF genes were identified in the M. truncatula genome. Protein domains and motifs of EFs were highly conserved in plants. MtEF1A1 has the highest expression levels in root nodules and roots, followed by the leaves and stems. Transgenic Arabidopsis thaliana overexpressing MtEF1A1 was more resistant to salt stress treatment, with higher germination rate, longer roots, and more lateral roots than wild type plant. In addition, lower levels of H2O2 and malondialdehyde (MDA) were also detected in transgenic Arabidopsis. Similarly, MtEF1A1 overexpressing M. truncatula was more resistant to salt stress and had lower levels of reactive oxygen species (ROS) in leaves. Furthermore, the expression levels of abiotic stress-responsive genes (MtRD22A and MtCOR15A) and calcium-binding genes (MtCaM and MtCBL4) were upregulated in MtEF1A1 overexpressing lines of M. truncatula. CONCLUSION: These results suggested that MtEF1A1 play a positive role in salt stress regulation. MtEF1A1 may realize its function by binding to calmodulin (CaM) or by participating in Ca2+-dependent signaling pathway. This study revealed that MtEF1A1 is an important regulator for salt stress response in M. truncatula, and provided potential strategy for salt-tolerant plant breeding.

Genome-wide identification and characterization of filamentation temperature-sensitive H (FtsH) genes and expression analysis in response to multiple stresses in Medicago truncatula.

BACKGROUND: Filamentation temperature-sensitive H (FtsH) is an AAA+ ATP-dependent protease that plays a vital role in plant environmental adaption and tolerance. However, little is known about the function of the FtsH gene family in the most important legume model plant, Medicago truncatula. METHODS AND RESULTS: To identify and investigate the potential stress adaptation roles of FtsH gene family in M. truncatula, we conducted a series of genome-wide characterization and expression analyses. Totally, twenty MtFtsH genes were identified, which were unevenly distributed across eight chromosomes and classified into six evolution groups based on their phylogenetic relationships, with each group containing similar structures and motifs. Furthermore, MtFtsH genes exhibited a high degree of collinearity and homology with leguminous plants such as alfalfa and soybean. Multiple cis-elements in the upstream region of MtFtsH genes were also identified that responded to light, abiotic stress, and phytohormones. Public RNA-seq data indicated that MtFtsH genes were induced under both salt and drought stresses, and our transcript expression analysis showed that MtFtsH genes of MtFtsH1, MtFtsH2, MtFtsH4, MtFtsH9, and MtFtsH10 were up-regulated after ABA, H2O2, PEG, and NaCl treatments. These results suggest that MtFtsH genes may play a critical role in drought and high salt stress responses and the adaption processes of plants. CONCLUSIONS: This study provides a systematic analysis of FtsH gene family in M. truncatula, serving as a valuable molecular theoretical basis for future functional investigations. Our findings also extend the pool of potential candidate genes for the genetic improvement of abiotic stress tolerance in legume crops.

Genome-Wide Analysis of the LATERAL ORGAN BOUNDARIES Domain (LBD) Members in Alfalfa and the Involvement of MsLBD48 in Nitrogen Assimilation.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a transcription factor family specific to the land plants, have been implicated in multiple biological processes including organ development, pathogen response and the uptake of inorganic nitrogen. The study focused on LBDs in legume forage Alfalfa. The genome-wide analysis revealed that in Alfalfa 178 loci across 31 allelic chromosomes encoded 48 unique LBDs (MsLBDs), and the genome of its diploid progenitor M. sativa spp. Caerulea encoded 46 LBDs. Synteny analysis indicated that the expansion of AlfalfaLBDs was attributed to the whole genome duplication event. The MsLBDs were divided into two major phylogenetic classes, and the LOB domain of the Class I members was highly conserved relative to that of the Class II. The transcriptomic data demonstrated that 87.5% of MsLBDs were expressed in at least one of the six test tissues, and Class II members were preferentially expressed in nodules. Moreover, the expression of Class II LBDs in roots was upregulated by the treatment of inorganic nitrogen such as KNO3 and NH4Cl (0.3 mM). The overexpression of MsLBD48, a Class II member, in Arabidopsis resulted in growth retardance with significantly declined biomass compared with the non-transgenic plants, and the transcription level of the genes involved in nitrogen uptake or assimilation, including NRT1.1, NRT2.1, NIA1 and NIA2 was repressed. Therefore, the LBDs in Alfalfa are highly conserved with their orthologs in embryophytes. Our observations that ectopic expression of MsLBD48 inhibited Arabidopsis growth by repressing nitrogen adaption suggest the negative role of the transcription factor in plant uptake of inorganic nitrogen. The findings imply the potential application of MsLBD48 in Alfalfa yield improvement via gene editing.

Genome-Wide Identification and Phylogenetic and Expression Analyses of the PLATZ Gene Family in Medicago sativa L.

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa, Zea mays, Triticum aestivum, Fagopyrum tataricum, and Arabidopsis thaliana; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes (MsPLATZs) were identified in the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZs have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs, and provides ideas for the improvement of alfalfa varieties and germplasm innovation.

Characterization of the Heat Shock Transcription Factor Family in Medicago sativa L. and Its Potential Roles in Response to Abiotic Stresses.

Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome ("Xinjiangdaye" reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT-PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa.

Transcriptome and GWAS Analyses Reveal Candidate Gene for Root Traits of Alfalfa during Germination under Salt Stress.

Alfalfa growth and production in China are negatively impacted by high salt concentrations in soils, especially in regions with limited water supplies. Few reliable genetic markers are currently available for salt tolerance selection. As a result, molecular breeding strategies targeting alfalfa are hindered. Therefore, with the continuous increase in soil salinity in agricultural lands, it is indispensable that a salt-tolerant variety of alfalfa is produced. We collected 220 alfalfa varieties around the world for resequencing and performed genome-wide association studies (GWASs). Alfalfa seeds were germinated in saline water with different concentrations of NaCl, and the phenotypic differences in several key root traits were recorded. In the phenotypic analysis, the breeding status and geographical origin strongly affected the salt tolerance of alfalfa. Forty-nine markers were significantly associated with salt tolerance, and 103 candidate genes were identified based on linkage disequilibrium. A total of 2712 differentially expressed genes were upregulated and 3570 were downregulated based on transcriptomic analyses. Some candidate genes that affected root development in the seed germination stage were identified through the combination of GWASs and transcriptome analyses. These genes could be used for molecular breeding strategies to increase alfalfa's salt tolerance and for further research on salt tolerance in general.

MLK4-mediated phosphorylation of histone H3T3 promotes flowering by transcriptional silencing of FLC/MAF in Arabidopsis thaliana.

Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9-LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with the transcriptional repression of euchromatic and heterochromatic loci. This study demonstrates that mlk4-3, a T-DNA insertion mutant of MLK4, flowered late, and that overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3T3 both in vitro and in vivo, and this catalytic activity required the conserved lysine residue K175. mutation of MLK4 at K175 failed to restore the level of phosphorylated H3T3 (H3T3ph) or to complement the phenotypic defects of mlk4-3. The FLC/MAF-clade genes, including FLC, MAF4 and MAF5, were significantly upregulated in mlk4-3. The double mutant mlk4-3 flc-3 flowered earlier than mlk4-3, suggesting that functional FLC is crucial for flowering repression in mlk4-3. Chromatin immunoprecipitation assays showed that MLK4 bound to FLC/MAF chromatin and that H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. In accordance, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4-3 but diminished in 35S::MLK4(K175R)/mlk4-3 plants. Moreover, the amount of RNA Pol II deposited at FLC/MAF was clearly enriched in mlk4-3 relative to the wild type. Therefore, MLK4-dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC/MAF. This study sheds light on the delicate control of flowering by the plant-specific CK1, MLK4, via post-translational modification of histone H3.

Genome-wide identification, phylogeny and expression analysis of the SPL gene family and its important role in salt stress in Medicago sativa L.

BACKGROUND: SQUAMOSA promoter-binding protein-like (SPL) transcription factors are widely present in plants and are involved in signal transduction, the stress response and development. The SPL gene family has been characterized in several model species, such as A. thaliana and G. max. However, there is no in-depth analysis of the SPL gene family in forage, especially alfalfa (Medicago sativa L.), one of the most important forage crops worldwide. RESULT: In total, 76 putative MsSPL genes were identified in the alfalfa genome with an uneven distribution. Based on their identity and gene structure, these MsSPLs were divided into eight phylogenetic groups. Seventy-three MsSPL gene pairs arose from segmental duplication events, and the MsSPLs on the four subgenomes of individual chromosomes displayed high collinearity with the corresponding M. truncatula genome. The prediction of the cis-elements in the promoter regions of the MsSPLs detected two copies of ABA (abscisic acid)-responsive elements (ABREs) on average, implying their potential involvement in alfalfa adaptation to adverse environments. The transcriptome sequencing of MsSPLs in roots and leaves revealed that 54 MsSPLs were expressed in both tissues. Upon salt treatment, three MsSPLs (MsSPL17, MsSPL23 and MsSPL36) were significantly regulated, and the transcription level of MsSPL36 in leaves was repressed to 46.6% of the control level. CONCLUSION: In this study, based on sequence homology, we identified 76 SPL genes in the alfalfa. The SPLs with high identity shared similar gene structures and motifs. In total, 71.1% (54 of 76) of the MsSPLs were expressed in both roots and leaves, and the majority (74.1%) preferred underground tissues to aerial tissues. MsSPL36 in leaves was significantly repressed under salt stress. These findings provide comprehensive information regarding the SPB-box gene family for improve alfalfa tolerance to high salinity.

Combining QTL mapping and RNA-Seq Unravels candidate genes for Alfalfa (Medicago sativa L.) leaf development.

BACKGROUND: Leaf size affects crop canopy morphology and photosynthetic efficiency, which can influence forage yield and quality. It is of great significance to mine the key genes controlling leaf development for breeding new alfalfa varieties. In this study, we mapped leaf length (LL), leaf width (LW), and leaf area (LA) in an F1 mapping population derived from a cultivar named ZhongmuNo.1 with larger leaf area and a landrace named Cangzhou with smaller leaf area. RESULTS: This study showed that the larger LW was more conducive to increasing LA. A total of 24 significant quantitative trait loci (QTL) associated with leaf size were identified on both the paternal and maternal linkage maps. Among them, nine QTL explained about 11.50-22.45% phenotypic variation. RNA-seq analysis identified 2,443 leaf-specific genes and 3,770 differentially expressed genes. Combining QTL mapping, RNA-seq alalysis, and qRT-PCR, we identified seven candidate genes associated with leaf development in five major QTL regions. CONCLUSION: Our study will provide a theoretical basis for marker-assisted breeding and lay a foundation for further revealing molecular mechanism of leaf development in alfalfa.

Identification and characterization of stress responsive homeodomain leucine zipper transcription factors in Medicago truncatula.

BACKGROUND: Homeodomain leucine zipper (HD-ZIP) transcription factors play roles in regulating plant development and responses to abiotic stresses; however, how HD-ZIP genes in Medicago truncatula are involved in abiotic stress response remains elusive. METHODS AND RESULTS: The HD-ZIP I genes in Medicago truncatula were identified and characterized, and their expression patterns in different tissues and under different abiotic stresses were analyzed. A total of 15 Medicago truncatula HD-ZIP I genes were identified and a phylogenetic analysis of HD-ZIP I proteins in Arabidopsis thaliana and Medicago truncatula was conducted. Fifteen HD-ZIP I genes showed diverse tissue preferences. Among them, expressions of MtHB22 and MtHB51 were specially detected in vegetative buds. In addition, they responded to various abiotic stresses, including salinity and osmotic stress and abscisic acid (ABA). For instance, MtHB7 and MtHB12 expression levels were found to be positively associated with salt, osmotic stress and ABA in both shoots and roots, while MtHB13 and MtHB23 were negatively associated with these stresses in Medicago truncatula. CONCLUSION: The HD-ZIP I genes in Medicago truncatula are evolutionarily conserved, but also exhibit gene duplication and gene loss events. Differential expression analysis of Medicago truncatula HD-ZIP I genes indicated their crucial roles in abiotic stress responses. Our genome-wide analysis of the HD-ZIP I transcription factor family in Medicago truncatula provided a valuable reference for further research.

Genome-Wide Identification and Expression Analysis of the NAC Gene Family in Alfalfa Revealed Its Potential Roles in Response to Multiple Abiotic Stresses.

NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa "Xinjiangdaye" reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT−qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa.

Overexpression of Mtr-miR319a Contributes to Leaf Curl and Salt Stress Adaptation in Arabidopsis thaliana and Medicago truncatula.

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.

Gibberellins Inhibit Flavonoid Biosynthesis and Promote Nitrogen Metabolism in Medicago truncatula.

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA3 and GA4) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA3. In contrast, nitrogen metabolism was promoted by exogenous GA3 but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.

A global alfalfa diversity panel reveals genomic selection signatures in Chinese varieties and genomic associations with root development.

Alfalfa (Medicago sativa L.) is an important forage crop worldwide. However, little is known about the effects of breeding status and different geographical populations on alfalfa improvement. Here, we sequenced 220 alfalfa core germplasms and determined that Chinese alfalfa cultivars form an independent group, as evidenced by comparisons of FST values between different subgroups, suggesting that geographical origin plays an important role in group differentiation. By tracing the influence of geographical regions on the genetic diversity of alfalfa varieties in China, we identified 350 common candidate genetic regions and 548 genes under selection. We also defined 165 loci associated with 24 important traits from genome-wide association studies. Of those, 17 genomic regions closely associated with a given phenotype were under selection, with the underlying haplotypes showing significant differences between subgroups of distinct geographical origins. Based on results from expression analysis and association mapping, we propose that 6-phosphogluconolactonase (MsPGL) and a gene encoding a protein with NHL domains (MsNHL) are critical candidate genes for root growth. In conclusion, our results provide valuable information for alfalfa improvement via molecular breeding.

De novo hydroponics system efficiency for the cuttings of alfalfa (Medicago sativa L.).

The legume plant alfalfa (Medicago sativa L.) is a widely cultivated perennial forage due to its high protein content, palatability, and strong adaptability to diverse agro-ecological zones. Alfalfa is a self-incompatible cross-pollinated autotetraploid species with tetrasomic inheritance. Therefore, maintaining excellent traits through seed reproduction is a prime challenge in alfalfa. However, the cutting propagation technology could enable consistent multiplication of quality plants that are genetically identical to the parent plant. The current study aimed to develop a simple, cost-effective, reproducible, and efficient hydroponic cutting method to preserve alfalfa plants and for molecular research. In this study, alfalfa landrace 'Wudi' was grown in hydroponics for 30 days and used as source material for cuttings. The top, middle and bottom sections of its stem were used as cuttings. The rooting rate, root length, and stem height of the different stem sections were compared to determine the best segment for alfalfa propagation in four nutrient treatments (HM, HM + 1/500H, HM + 1/1000H and d HM + 1/2000H). After 21 days of culture, the rooting rates of all the three stem types under four cutting nutrient solutions were above 78%. The rooting rate of the middle and bottom parts in HM + 1/1000 H and HM + 1/2000 H nutrient solutions reached more than 93%, with a higher health survey score (> 4.70). In conclusion, this study developed a de novo cutting propagation method that can be used to conserve and propagate germplasm in breeding programs and research. This method is a new report on the cutting propagation of alfalfa by hydroponics, which could supplement the existing cutting propagation methods.

iTRAQ-based comparative proteomic analysis of differences in the protein profiles of stems and leaves from two alfalfa genotypes.

BACKGROUND: To explore the molecular regulatory mechanisms of early stem and leaf development, proteomic analysis was performed on leaves and stems of F genotype alfalfa, with thin stems and small leaves, and M genotype alfalfa, with thick stems and large leaves. RESULTS: Based on fold-change thresholds of > 1.20 or < 0.83 (p < 0.05), a large number of proteins were identified as being differentially enriched between the M and F genotypes: 249 downregulated and 139 upregulated in stems and 164 downregulated and 134 upregulated in leaves. The differentially enriched proteins in stems were mainly involved in amino acid biosynthesis, phenylpropanoid biosynthesis, carbon fixation, and phenylalanine metabolism. The differentially enriched proteins in leaves were mainly involved in porphyrin and chlorophyll metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism, and carbon fixation in photosynthetic organisms. Six differentially enriched proteins were mapped onto the porphyrin and chlorophyll metabolism pathway in leaves of the M genotype, including five upregulated proteins involved in chlorophyll biosynthesis and one downregulated protein involved in chlorophyll degradation. Eleven differentially enriched proteins were mapped onto the phenylpropanoid pathway in stems of the M genotype, including two upregulated proteins and nine downregulated proteins. CONCLUSION: Enhanced chlorophyll synthesis and decreased lignin synthesis provided a reasonable explanation for the larger leaves and lower levels of stem lignification in M genotype alfalfa. This proteomic study aimed to classify the functions of differentially enriched proteins and to provide information on the molecular regulatory networks involved in stem and leaf development.

Up-regulation of lipid metabolism and glycine betaine synthesis are associated with choline-induced salt tolerance in halophytic seashore paspalum.

Choline may affect salt tolerance by regulating lipid and glycine betaine (GB) metabolism. This study was conducted to determine whether alteration of lipid profiles and GB metabolism may contribute to choline regulation and genotypic variations in salt tolerance in a halophytic grass, seashore paspalum (Paspalum vaginatum). Plants of Adalayd and Sea Isle 2000 were subjected to salt stress (200-mM NaCl) with or without foliar application of choline chloride (1 mM). Genotypic variations in salt tolerance and promotive effects of choline application on salt tolerance were associated with both the up-regulation of lipid metabolism and GB synthesis. The genotypic variations in salt tolerance associated with lipid metabolism were reflected by the differential accumulation of phosphatidylcholine and phosphatidylethanolamine between Adalayd and Sea Isle 2000. Choline-induced salt tolerance was associated with of the increase in digalactosyl diacylglycerol (DGDG) content including DGDG (36:4 and 36:6) in both cultivars of seashore paspalum and enhanced synthesis of phosphatidylinositol (34:2, 36:5, and 36:2) and phosphatidic acid (34:2, 34:1, and 36:5), as well as increases in the ratio of digalactosyl diacylglycerol: monogalactosyl diacylglycerol (DGDG:MGDG) in salt-tolerant Sea Isle 2000. Choline regulation of salt tolerance may be due to the alteration in lipid metabolism in this halophytic grass species.

Genome-wide AP2/ERF gene family analysis reveals the classification, structure, expression profiles and potential function in orchardgrass (Dactylis glomerata).

The AP2/ERF transcription factor (TF) family is of great importance in developmental regulation and responses to stress and pathogenic stimuli. Orchardgrass (Dactylis glomerata), a perennial cold-season forage of high quality in the world's temperate zones, contributes to grazing land through mixed sowing with alfalfa (Medicago sativa) and white clover (Trifolium repens). However, little is known about AP2/ERF TFs in orchardgrass. In this study, 193 AP2/ERF genes were classified into five subfamilies and 13 subgroups through phylogenetic analysis. Chromosome structure analysis showed that AP2/ERF family genes in orchardgrass were distributed on seven chromosomes and specific conservative sequences were found in each subgroup. Exon-intron structure and motifs in the same subgroup were almost identical, and the unique motifs contributed to the classification and functional annotation of DgERFs. Expression analysis showed tissue-specific expression of DgERFs in roots and flowers, with most DgERFs widely expressed in roots. The expression levels of each subgroup (subgroups Vc, VIIa, VIIIb, IXa, and XIa) were high at the before-heading and heading stages (BH_DON and H_DON). In addition, 12 DgERFs in various tissues and five DgERFs associated with abiotic stresses were selected for qRT-PCR analysis showed that four dehydration-responsive element binding (DREB) genes and one ERF subfamily gene in orchardgrass were regulated with PEG, heat and salt stresses. DgERF056 belonged to ERF subfamily was involved in the processes of flowering and development stage. This study systematic explored the DgERFs at the genome level for the first time, which lays a foundation for a better understanding of AP2/ERF gene function in Dactylis glomerata and other types of forage.