A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the ER exit sites (ERESs) and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12 and Sec24, and participates in coat protein II (COPII) vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/ phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER, and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

GA dynamics governing nodulation revealed using GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula lateral organs.

During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Genetic basis of the historical iron-accumulating dgl and brz mutants in pea.

The Pisum sativum (pea) mutants degenerate leaves (dgl) and bronze (brz) accumulate large amounts of iron in leaves. First described several decades ago, the two mutants have provided important insights into iron homeostasis in plants but the underlying mutations have remained unknown. Using exome sequencing we identified an in-frame deletion associated with dgl in a BRUTUS homolog. The deletion is absent from wild type and the original parent line. BRUTUS belongs to a small family of E3 ubiquitin ligases acting as negative regulators of iron uptake in plants. The brz mutation was previously mapped to chromosome 4, and superimposing this region to the pea genome sequence uncovered a mutation in OPT3, encoding an oligopeptide transporter with a plant-specific role in metal transport. The causal nature of the mutations was confirmed by additional genetic analyses. Identification of the mutated genes rationalizes many of the previously described phenotypes and provides new insights into shoot-to-root signaling of iron deficiency. Furthermore, the non-lethal mutations in these essential genes suggest new strategies for biofortification of crops with iron.

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat.

Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB-bHLH-WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain-leucine zipper (HD-ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production.

Genome-wide methylation landscape during somatic embryogenesis in Medicago truncatula reveals correlation between Tnt1 retrotransposition and hyperactive methylation regions.

Medicago truncatula is a model legume for fundamental research on legume biology and symbiotic nitrogen fixation. Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants in M. truncatula R108. Approximately 21 000 insertion lines have been generated and publicly available. Tnt1 retro-transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE of M. truncatula R108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2-kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site of Tnt1 insertions in M. truncatula R108 and stronger hypermethylation of genes correlated with higher number of Tnt1 insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and the Tnt1 retrotransposition correlates with the hyperactive methylation regions.

MtPIN4 plays critical roles in amino acid biosynthesis and metabolism of seed in Medicago truncatula.

The regulation of seed development is critical for determining crop yield. Auxins are vital phytohormones that play roles in various aspects of plant growth and development. However, its role in amino acid biosynthesis and metabolism in seeds is not fully understood. In this study, we identified a mutant with small seeds through forward genetic screening in Medicago truncatula. The mutated gene encodes MtPIN4, an ortholog of PIN1. Using molecular approaches and integrative omics analyses, we discovered that auxin and amino acid content significantly decreased in mtpin4 seeds, highlighting the role of MtPIN4-mediated auxin distribution in amino acid biosynthesis and metabolism. Furthermore, genetic analysis revealed that the three orthologs of PIN1 have specific and overlapping functions in various developmental processes in M. truncatula. Our findings emphasize the significance of MtPIN4 in seed development and offer insights into the molecular mechanisms governing the regulation of seed size in crops. This knowledge could be applied to enhance crop quality by targeted manipulation of seed protein regulatory pathways.

Metabolomics and transcriptomics analysis revealed the response mechanism of alfalfa to combined cold and saline-alkali stress.

Cold and saline-alkali stress are frequently encountered by plants, and they often occur simultaneously in saline-alkali soils at mid to high latitudes, constraining forage crop distribution and production. However, the mechanisms by which forage crops respond to the combination of cold and saline-alkali stress remain unknown. Alfalfa (Medicago sativa L.) is one of the most essential forage grasses in the world. In this study, we analyzed the complex response mechanisms of two alfalfa species (Zhaodong [ZD] and Blue Moon [BM]) to combined cold and saline-alkali stress using multi-omics. The results revealed that ZD had a greater ability to tolerate combined stress than BM. The tricarboxylic acid cycles of the two varieties responded positively to the combined stress, with ZD accumulating more sugars, amino acids, and jasmonic acid. The gene expression and flavonoid content of the flavonoid biosynthesis pathway were significantly different between the two varieties. Weighted gene co-expression network analysis and co-expression network analysis based on RNA-Seq data suggested that the MsMYB12 gene may respond to combined stress by regulating the flavonoid biosynthesis pathway. MsMYB12 can directly bind to the promoter of MsFLS13 and promote its expression. Moreover, MsFLS13 overexpression can enhance flavonol accumulation and antioxidant capacity, which can improve combined stress tolerance. These findings provide new insights into improving alfalfa resistance to combined cold and saline-alkali stress, showing that flavonoids are essential for plant resistance to combined stresses, and provide theoretical guidance for future breeding programs.

Paired Medicago receptors mediate broad-spectrum resistance to nodulation by Sinorhizobium meliloti carrying a species-specific gene.

Plants have evolved the ability to distinguish between symbiotic and pathogenic microbial signals. However, potentially cooperative plant-microbe interactions often abort due to incompatible signaling. The Nodulation Specificity 1 (NS1) locus in the legume Medicago truncatula blocks tissue invasion and root nodule induction by many strains of the nitrogen-fixing symbiont Sinorhizobium meliloti. Controlling this strain-specific nodulation blockade are two genes at the NS1 locus, designated NS1a and NS1b, which encode malectin-like leucine-rich repeat receptor kinases. Expression of NS1a and NS1b is induced upon inoculation by both compatible and incompatible Sinorhizobium strains and is dependent on host perception of bacterial nodulation (Nod) factors. Both presence/absence and sequence polymorphisms of the paired receptors contribute to the evolution and functional diversification of the NS1 locus. A bacterial gene, designated rns1, is required for activation of NS1-mediated nodulation restriction. rns1 encodes a type I-secreted protein and is present in approximately 50% of the nearly 250 sequenced S. meliloti strains but not found in over 60 sequenced strains from the closely related species Sinorhizobium medicae. S. meliloti strains lacking functional rns1 are able to evade NS1-mediated nodulation blockade.

Intracellular infection by symbiotic bacteria requires the mitotic kinase AURORA1.

The subcellular events occurring in cells of legume plants as they form transcellular symbiotic-infection structures have been compared with those occurring in premitotic cells. Here, we demonstrate that Aurora kinase 1 (AUR1), a highly conserved mitotic regulator, is required for intracellular infection by rhizobia in Medicago truncatula. AUR1 interacts with microtubule-associated proteins of the TPXL and MAP65 families, which, respectively, activate and are phosphorylated by AUR1, and localizes with them within preinfection structures. MYB3R1, a rhizobia-induced mitotic transcription factor, directly regulates AUR1 through two closely spaced, mitosis-specific activator cis elements. Our data are consistent with a model in which the MYB3R1-AUR1 regulatory module serves to properly orient preinfection structures to direct the transcellular deposition of cell wall material for the growing infection thread, analogous to its role in cell plate formation. Our findings indicate that the eukaryotically conserved MYB3R1-TPXL-AUR1-MAP65 mitotic module was conscripted to support endosymbiotic infection in legumes.

The Extraordinary Alkali Bee, Nomia melanderi (Halictidae), the World's Only Intensively Managed Ground-Nesting Bee.

Among the ground-nesting bees are several proven crop pollinators, but only the alkali bee (Nomia melanderi) has been successfully managed. In <80 years, it has become the world's most intensely studied ground-nesting solitary bee. In many ways, the bee seems paradoxical. It nests during the torrid, parched midsummer amid arid valleys and basins of the western United States, yet it wants damp nesting soil. In these basins, extensive monocultures of an irrigated Eurasian crop plant, alfalfa (lucerne), subsidize millions of alkali bees. Elsewhere, its polylectic habits and long foraging range allow it to stray into neighboring crops contaminated with insecticides. Primary wild floral hosts are either non-native or poorly known. Kleptoparasitic bees plague most ground nesters, but not alkali bees, which do, however, host other well-studied parasitoids. Building effective nesting beds requires understanding the hydraulic conductivity of silty nesting soils and its important interplay with specific soil mineral salts. Surprisingly, some isolated populations endure inhospitably cold climates by nesting amid hot springs. Despite the peculiarities and challenges associated with its management, the alkali bee remains the second most valuable managed solitary bee for US agriculture and perhaps the world.

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-wide identification of SHMT family genes in alfalfa (Medicago sativa) and its functional analyses under various abiotic stresses.

BACKGROUND: Alfalfa (Medicago sativa L.) is the most widely planted legume forage and one of the most economically valuable crops in the world. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays crucial roles in plant growth, development, and stress responses. To date, there has been no comprehensive bioinformatics investigation conducted on the SHMT genes in M. sativa. RESULTS: Here, we systematically analyzed the phylogenetic relationship, expansion pattern, gene structure, cis-acting elements, and expression profile of the MsSHMT family genes. The result showed that a total of 15 SHMT members were identified from the M. sativa genome database. Phylogenetic analysis demonstrated that the MsSHMTs can be divided into 4 subgroups and conserved with other plant homologues. Gene structure analysis found that the exons of MsSHMTs ranges from 3 to 15. Analysis of cis-acting elements found that each of the MsSHMT genes contained different kinds of hormones and stress-related cis-acting elements in their promoter regions. Expression and function analysis revealed that MsSHMTs expressed in all plant tissues. qRT-PCR analysis showed that MsSHMTs induced by ABA, Salt, and drought stresses. CONCLUSIONS: These results provided definite evidence that MsSHMTs might involve in growth, development and adversity responses in M. sativa, which laid a foundation for future functional studies of MsSHMTs.

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.

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.

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.

Biochar enhances the growth and physiological characteristics of Medicago sativa, Amaranthus caudatus and Zea mays in saline soils.

Biochar is a promising solution to alleviate the negative impacts of salinity stress on agricultural production. Biochar derived from food waste effect was investigated on three plant species, Medicago sativa, Amaranthus caudatus, and Zea mays, under saline environments. The results showed that biochar improved significantly the height by 30%, fresh weight of shoot by 35% and root by 45% of all three species compared to control (saline soil without biochar adding), as well as enhanced their photosynthetic pigments and enzyme activities in soil. This positive effect varied significantly between the 3 plants highlighting the importance of the plant-biochar interactions. Thus, the application of biochar is a promising solution to enhance the growth, root morphology, and physiological characteristics of plants under salt-induced stress.

The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza.

BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

Transcriptome analysis of resistant and susceptible Medicago truncatula genotypes in response to spring black stem and leaf spot disease.

Ascochyta blights cause yield losses in all major legume crops. Spring black stem (SBS) and leaf spot disease is a major foliar disease of Medicago truncatula and Medicago sativa (alfalfa) caused by the necrotrophic fungus Ascochyta medicaginicola. This present study sought to identify candidate genes for SBS disease resistance for future functional validation. We employed RNA-seq to profile the transcriptomes of a resistant (HM078) and susceptible (A17) genotype of M. truncatula at 24, 48, and 72 h post inoculation. Preliminary microscopic examination showed reduced pathogen growth on the resistant genotype. In total, 192 and 2,908 differentially expressed genes (DEGs) were observed in the resistant and susceptible genotype, respectively. Functional enrichment analysis revealed the susceptible genotype engaged in processes in the cell periphery and plasma membrane, as well as flavonoid biosynthesis whereas the resistant genotype utilized calcium ion binding, cell wall modifications, and external encapsulating structures. Candidate genes for disease resistance were selected based on the following criteria; among the top ten upregulated or downregulated genes in the resistant genotype, upregulated over time in the resistant genotype, hormone pathway genes, plant disease resistance genes, receptor-like kinases, contrasting expression profiles in QTL for disease resistance, and upregulated genes in enriched pathways. Overall, 22 candidate genes for SBS disease resistance were identified with support from the literature. These genes will be sources for future targeted mutagenesis and candidate gene validation potentially helping to improve disease resistance to this devastating foliar pathogen.