The long noncoding RNA LAL contributes to salinity tolerance by modulating LHCB1s' expression in Medicago truncatula.

Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s' expression in M. truncatula.

Physiological and lipidomic response of exogenous choline chloride alleviating salt stress injury in Kentucky bluegrass (Poa pratensis).

Introduction: Choline participates in plant stress tolerance through glycine betaine (GB) and phospholipid metabolism. As a salt-sensitive turfgrass species, Kentucky bluegrass (Poa pratensis) is the main turfgrass species in cool-season areas. Methods: To improve salinity tolerance and investigate the effects of choline on the physiological and lipidomic responses of turfgrass plants under salinity stress conditions, exogenous choline chloride was applied to Kentucky bluegrass exposed to salt stress. Results: From physiological indicators, exogenous choline chloride could alleviate salt stress injury in Kentucky bluegrass. Lipid analysis showed that exogenous choline chloride under salt-stress conditions remodeled the content of phospholipids, glycolipids, and lysophospholipids. Monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine content were increased and phosphatidic acid content were decreased in plants after exogenous choline chloride under salt treatment. Plant leaf choline content increased, but GB was not detected in exogenous choline chloride treatment plants under nonstress or salt-stress conditions. Discussion: GB synthesis pathway related genes showed no clear change to choline chloride treatment, whereas cytidyldiphosphate-choline (CDP-choline) pathway genes were upregulated by choline chloride treatment. These results reveal that lipid remodeling through choline metabolism plays an important role in the salt tolerance mechanism of Kentucky bluegrass. Furthermore, the lipids selected in this study could serve as biomarkers for further improvement of salt-sensitive grass species.

The role of Class â ¡ KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXII) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (orthologue of Arabidopsis Class M KNOX) and NO APICAL MERISTEM (orthologue of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for MtKNOX4-mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula.

Genome-Wide Identification and Characterization of the Msr Gene Family in Alfalfa under Abiotic Stress.

Alfalfa (Medicago sativa) is an important leguminous forage, known as the "The Queen of Forages". Abiotic stress seriously limits the growth and development of alfalfa, and improving the yield and quality has become an important research area. However, little is known about the Msr (methionine sulfoxide reductase) gene family in alfalfa. In this study, 15 Msr genes were identified through examining the genome of the alfalfa "Xinjiang DaYe". The MsMsr genes differ in gene structure and conserved protein motifs. Many cis-acting regulatory elements related to the stress response were found in the promoter regions of these genes. In addition, a transcriptional analysis and qRT-PCR (quantitative reverse transcription PCR) showed that MsMsr genes show expression changes in response to abiotic stress in various tissues. Overall, our results suggest that MsMsr genes play an important role in the response to abiotic stress for alfalfa.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Evaluation of the tolerance and forage quality of different ecotypes of seashore paspalum.

Seashore paspalum is a halophytic, warm-season grass with wide applications. It is noted for its superior salt tolerance in saline environments; however, the nutritive value of seashore paspalum and the effect of salinity remains to be determined. Therefore, this study aimed to evaluate the relationship between agronomic traits and forage quality and identified the effects of short-term high-salt stress (1 week, 700 mM NaCl) on the growth and forage nutritive value of 16 ecotypes of seashore paspalum. The salt and cold tolerances of the seashore paspalum ecotypes were assessed based on the survival rate following long-term high-salt stress (7 weeks, 700 mM NaCl) and exposure to natural low temperature stress. There were significant genetic (ecotype-specific) effects on plant height, leaf-stem ratio, and survival rate of seashore paspalum following salt or low temperature stress. Plant height was significantly negatively correlated with the leaf-stem ratio (r = -0.63, P<0.01), but the heights and leaf-stem ratios were not significantly correlated with the fresh weight (FW) and dry weight (DW) of the shoots. High salinity decreased the FW and DW of the shoots by 50.6% and 23.6%, respectively, on average. Seashore paspalum exhibited outstanding salt tolerance and forage quality at high salinity. The survival rate of the different ecotypes of seashore paspalum varied from 6.5% to 49.0% following treatment with 700 mM NaCl for 7 weeks. The crude protein (CP) content of the control and treatment groups (700 mM NaCl) was 17.4% and 19.3%, respectively, of the DW on average, and the CP content of most ecotypes was not significantly influenced by high salinity. The average ether extract (EE) content ranged from 4.6% to 4.4% of the DW under control and saline conditions, respectively, indicating that the influence was not significant. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of the control group were 57.4% and 29.8%, respectively, of the DW on average. Salt stress reduced the content of NDF and ADF to 50.2% and 25.9%, respectively, of the DW on average. Altogether, the results demonstrated that stress did not have any significant effects on the CP and EE content of most ecotypes, but reduced the NDF and ADF content and improved relative feed value (RFV). The results obtained herein support the notion that seashore paspalum is a good candidate for improving the forage potential of saline soils and can provide useful guidelines for livestock producers.

Genome-wide identification of growth-regulating factor transcription factor family related to leaf and stem development in alfalfa.

Growth-regulating factors (GRFs) play crucial roles in plant growth and stress response. To date, there have been no reports of the analysis and identification of the GRF transcription factor family in alfalfa. In this study, we identified 27 GRF family members from alfalfa (Medicago sativa L.) "Xinjiang Daye", and analyzed their physicochemical properties. Based on phylogenetic analysis, these MsGRFs were divided into five subgroups, each with a similar gene structure and conserved motifs. MsGRFs genes are distributed on 23 chromosomes, and all contain QLQ and WRC conserved domains. The results of the collinearity analysis showed that all MsGRFs are involved in gene duplication, including multiple whole-genome duplication or segmental duplication and a set of tandem duplication, indicating that large-scale duplication is important for the expansion of the GRF family in alfalfa. Several hormone-related and stress-related cis-acting elements have been found in the promoter regions of MsGRFs. Some MsGRFs were highly expressed in young leaves and stems, and their expression decreased during development. In addition, the leaf size of different varieties was found to vary, and MsGRF1 to 4, MsGRF18 to 20, and MsGRF22 to 23 were differentially expressed in large and small leaf alfalfa varieties, suggesting that they are critical in the regulation of leaf size. The results of this study can benefit further exploration of the regulatory functions of MsGRFs in growth and development, and can identify candidate genes that control leaf size development.

Effects of autotoxicity and allelopathy on seed germination and seedling growth in Medicago truncatula.

Autotoxicity is a form of intraspecific allelopathy, in which a plant species inhibits the establishment or growth of the same species through the release of toxic chemical compounds into the environment. The phenomenon of autotoxicity in crops is best traced in alfalfa (Medicago sativa). A close relative of alfalfa, M. truncatula, has been developed into an excellent model species for leguminous plants. However, it is not known whether M. truncatula has autotoxicity. In this study, M. truncatula root exudates showed a negative impact on the growth of M. truncatula seedlings, indicating autotoxicity. Detailed analyses with plant extracts from M. truncatula and alfalfa revealed varying degrees of suppression effects in the two species. The extracts negatively affected seed germination potential, germination rate, radicle length, hypocotyl length, synthetic allelopathic effect index, plant height, root growth, fresh weight, dry weight, net photosynthetic rate, transpiration rate, and stomatal conductance in both M. truncatula and alfalfa. The results demonstrated that autotoxicity and allelopathic effects exist in M. truncatula. This opens up a new way to use M. truncatula as a model species to carry out in-depth studies of autotoxicity and allelopathy to elucidate biochemical pathways of allelochemicals and molecular networks controlling biosynthesis of the chemicals.

Comprehensive transcriptomic and metabolomic profiling reveals the differences between alfalfa sprouts germinated with or without light exposure.

Alfalfa sprouts are among the most nutritionally rich foods, and light exposure is a critical factor in determining their biomass and quality. However, detailed metabolic and molecular differences between yellow and green alfalfa sprouts remain unclear. In this study, comprehensive metabolomic and transcriptomic analyses were integrated to evaluate the nutrient composition of alfalfa sprouts during germination with or without light exposure. Differentially expressed genes and differentially accumulated metabolites in green and yellow alfalfa sprouts were significantly enriched in secondary metabolic pathways, such as the isoflavonoid biosynthesis pathway. Green alfalfa sprouts contained a wide variety of lipids, flavonoids, phenolic acids, and terpenoids, among which the top three upregulated were calycosin, methyl gallate, and epicatechin 3-gallate, whereas yellow alfalfa sprouts contained relatively more isoquercitrin. These results provide new insights into the nutritional value and composition of alfalfa sprouts under different germination regimes.

The formation of stipule requires the coordinated actions of the legume orthologs of Arabidopsis BLADE-ON-PETIOLE and LEAFY.

Stipule morphology is a classical botanical key character used in plant identification. Stipules are considerably diverse in size, function and architecture, such as leaf-like stipules, spines or tendrils. However, the molecular mechanism that regulates stipule identity remains largely unknown. We isolated mutants with abnormal stipules. The mutated gene encodes the NODULE ROOT1 (MtNOOT1), which is the ortholog of BLADE-ON-PETIOLE (BOP) in Medicago truncatula. We also obtained mutants of MtNOOT2, the homolog of MtNOOT1, but they do not show obvious defects in stipules. The mtnoot1 mtnoot2 double mutant shows a higher proportion of transformation from stipules to leaflet-like stipules than the single mutants, suggesting that they redundantly determine stipule identity. Further investigations show that MtNOOTs control stipule initiation together with SINGLE LEAFLET1 (SGL1), which functions in development of lateral leaflets. Increasing SGL1 activity in mtnoot1 mtnoot2 is sufficient for the transformation of stipules to leaves. Moreover, MtNOOTs inhibit SGL1 expression during stipule development, which is probably conserved in legume species. Our study proposes a genetic regulatory model for stipule development, specifically with regard to the MtNOOTs-SGL1 module, which functions in two phases of stipule development, first in the control of stipule initiation and second in stipule patterning.

Transcriptome Analysis of Leaf Senescence Regulation Under Alkaline Stress in Medicago truncatula.

In plants, the leaf is an essential photosynthetic organ, and is the primary harvest in forage crops such as alfalfa (Medicago sativa). Premature leaf senescence caused by environmental stress can result in significant yield loss and quality reduction. Therefore, the stay-green trait is important for improving the economic value of forage crops. Alkaline stress can severely damage leaf cells and, consequently, cause leaf senescence. To understand the molecular regulatory mechanisms and identify vital senescence-associated genes under alkaline stress, we used high-throughput sequencing to study transcriptional changes in Medicago truncatula, a model plant for forage crops. We identified 2,165 differentially expressed genes, 985 of which were identical to those in the dark-induced leaf senescence group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 985 genes were mainly enriched in nutrient cycling processes such as cellular amino acid metabolic processes and organic substance catabolic processes, indicating nutrient redistribution. The other 1,180 differentially expressed genes were significantly enriched in the oxidoreductase complex, aerobic respiration, and ion transport. Our analysis showed the two gene sets guiding the coupled physiological and biochemical alterations play different roles under alkaline stress with a coordinated and integrated way. Many transcription factor families were identified from these differentially expressed genes, including MYB, WRKY, bHLH, and NAC which have particular preference involved in stress resistance and regulation of senescence. Our results contribute to the exploration of the molecular regulatory mechanisms of leaf senescence in M. truncatula under alkaline stress and provide new candidate genes for future breeding to improve the biomass and quality of forage crops.

Protoplast Isolation, Transformation, and Regeneration for Forage and Turf Grasses.

Forage and turf grasses are widely grown and contribute significantly to sustainable agriculture. This chapter describes a protocol for protoplast transformation and plant regeneration for major forage and turf grass species, including tall fescue, red fescue, meadow fescue, perennial ryegrass, and Italian ryegrass. Embryogenic calli induced from caryopsis were used to establish embryogenic cell suspension cultures. Protoplasts were isolated from embryogenic suspension cultures and used for direct gene transfer. Chimeric genes were introduced into protoplasts by polyethylene glycol treatment. Upon selection with antibiotics or herbicide, resistant calli were obtained and transgenic plants were regenerated from these calli.

The CCCH zinc finger protein C3H15 negatively regulates cell elongation by inhibiting brassinosteroid signaling.

Plant CCCH proteins participate in the control of multiple developmental and adaptive processes, but the regulatory mechanisms underlying these processes are not well known. In this study, we showed that the Arabidopsis (Arabidopsis thaliana) CCCH protein C3H15 negatively regulates cell elongation by inhibiting brassinosteroid (BR) signaling. Genetic and biochemical evidence showed that C3H15 functions downstream of the receptor BR INSENSITIVE 1 (BRI1) as a negative regulator in the BR pathway. C3H15 is phosphorylated by the GLYCOGEN SYNTHASE KINASE 3 -like kinase BR-INSENSITIVE 2 (BIN2) at Ser111 in the cytoplasm in the absence of BRs. Upon BR perception, C3H15 transcription is enhanced, and the phosphorylation of C3H15 by BIN2 is reduced. The dephosphorylated C3H15 protein accumulates in the nucleus, where C3H15 regulates transcription via G-rich elements (typically GGGAGA). C3H15 and BRASSINAZOLE RESISTANT 1 (BZR1)/BRI1-EMS-SUPPRESSOR 1 (BES1), two central transcriptional regulators of BR signaling, directly suppress each other and share a number of BR-responsive target genes. Moreover, C3H15 antagonizes BZR1 and BES1 to regulate the expression of their shared cell elongation-associated target gene, SMALL AUXIN-UP RNA 15 (SAUR15). This study demonstrates that C3H15-mediated BR signaling may be parallel to, or even attenuate, the dominant BZR1 and BES1 signaling pathways to control cell elongation. This finding expands our understanding of the regulatory mechanisms underlying BR-induced cell elongation in plants.

Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor-like kinase.

Soil-borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole-genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor-like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor-root colonization by the ectomycorrhizal fungus Laccaria bicolor. By introducing PtLecRLK1 into a perennial grass known to be a non-host of L. bicolor, switchgrass (Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro-PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non-host to a host of L. bicolor. Furthermore, transcriptomic and proteomic analyses on inoculated-transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down-regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N-containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non-host to enhance plant productivity and fitness on marginal lands.

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.

Comparative Transcriptome Analysis of Salt Stress-Induced Leaf Senescence in Medicago truncatula.

Leaves are the most critical portion of forage crops such as alfalfa (Medicago sativa). Leaf senescence caused by environmental stresses significantly impacts the biomass and quality of forages. To understand the molecular mechanisms and identify the key regulator of the salt stress-induced leaf senescence process, we conducted a simple and effective salt stress-induced leaf senescence assay in Medicago truncatula, which was followed by RNA-Seq analysis coupled with physiological and biochemical characterization. By comparing the observed expression data with that derived from dark-induced leaf senescence at different time points, we identified 3,001, 3,787, and 4,419 senescence-associated genes (SAGs) for salt stress-induced leaf senescence on day 2, 4, and 6, respectively. There were 1546 SAGs shared by dark and salt stress treatment across the three time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 1546 SAGs were mainly related to protein and amino acids metabolism, photosynthesis, chlorophyll metabolism, and hormone signaling during leaf senescence. Strikingly, many different transcription factors (TFs) families out of the 1546 SAGs, including NAC, bHLH, MYB, and ERF, were associated with salt stress-induced leaf senescence processes. Using the transient expression system in Nicotiana benthamiana, we verified that three functional NAC TF genes from the 1546 SAGs were related to leaf senescence. These results clarify SAGs under salt stress in M. truncatula and provide new insights and additional genetic resources for further forage crop breeding.

Identification of a gene responsible for seedpod spine formation and other phenotypic alterations using whole-genome sequencing analysis in Medicago truncatula.

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.

A seed coat-specific ß-ketoacyl-CoA synthase, KCS12, is critical for preserving seed physical dormancy.

Physical dormancy in seeds exists widely in seed plants and plays a vital role in maintaining natural seed banks. The outermost cuticle of the seed coat forms a water-impermeable layer, which is critical for establishing seed physical dormancy. We previously set up the legume plant Medicago truncatula as an excellent model for studying seed physical dormancy, and our studies revealed that a class II KNOTTED-like homeobox, KNOX4, is a transcription factor critical for controlling hardseededness. Here we report the function of a seed coat ß-ketoacyl-CoA synthase, KCS12. The expression level of KCS12 is significantly downregulated in the knox4 mutant. The KCS12 gene is predominantly expressed in the seed coat, and seed development in the M. truncatula kcs12 mutant is altered. Further investigation demonstrated that kcs12 mutant seeds lost physical dormancy and were able to absorb water without scarification treatment. Chemical analysis revealed that concentrations of C24:0 lipid polyester monomers are significantly decreased in mutant seeds, indicating that KCS12 is an enzyme that controls the production of very long chain lipid species in the seed coat. A chromatin immunoprecipitation assay demonstrated that the expression of KCS12 in the seed coat is directly regulated by the KNOX4 transcription factor. These findings define a molecular mechanism by which KNOX4 and KCS12 control formation of the seed coat and seed physical dormancy.