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Silicon (Si) supplementation can enhance symbiotic functions in some legumes (Fabaceae) with their nitrogen‐fixing rhizobia, such as root nodulation and nitrogen fixation. However, it is still poorly understood how Si influences legume–insect interactions. Here, we investigated how a symbiotic legume responds not only to Si supplementation but also to herbivory treatment with varying infestation levels in two events.We conducted a controlled climate chamber experiment by growing Medicago truncatula plants inoculated with rhizobia. For half of the plants, the soil was kept without Si (−Si), whereas the other half was regularly supplemented with Si (+Si). We then infested the plants with caterpillars of Spodoptera littoralis with 0, 1 or 3 larvae and 0, 1 or 1 larva in single herbivory attack and in double herbivory attack, respectively. To understand plant responses to such treatment combinations, we examined 16 functional traits.Nodule number, nodule fresh mass and nodule leghaemoglobin concentrations were not affected in single attack plants. However, increasing levels of herbivory led to decreases in such measured traits in double attack plants. Foliar C to N ratio increased in single attack plants but decreased in double attack plants with increasing levels of herbivory, indicating contrasting resource allocation. Herbivory did not affect the content of foliar Si, which was higher in +Si than −Si plants. Si and herbivory led to reduced foliar phenolics in double attack plants, suggesting a potential trade‐off between silicification and phenolic production. Si and herbivory led to increased trichome densities in single attack plants, but patterns were less clear in double attack plants. Herbivory but not Si reduced plant biomass with increasing levels of herbivory in double attack plants. Relative growth rates of the caterpillars, as proxy for plant resistance, decreased mainly due to herbivory treatment, when fed on single attack plants.Using a trait‐based approach, we provide novel insights to better understand the response of a legume to Si supplementation and different herbivory levels and events. We conclude that herbivory predominantly exerts much stronger effects than Si on various plant traits, pointing to a necessity to respond to herbivory by induced defence strategies. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides are systemic regulators of legume-rhizobium symbiosis that negatively control the number of nitrogen-fixing nodules. In Medicago truncatula, the expression of the MtCLE35 gene is activated in response to rhizobial inoculation and nitrate treatment, and its overexpression systemically inhibits nodulation. However, little is known about the molecular mechanisms underlying MtCLE35-mediated inhibition of nodulation. In order to elucidate target genes regulated by the MtCLE35-induced signaling cascade, we analyzed the transcriptome of MtCLE35-overexpressing roots inoculated by rhizobia using MACE (Massive Analysis of cDNA Ends) sequencing. Totally, 1390 genes were found to be differentially expressed between MtCLE35-overexpressing (35S::MtCLE35) and control (35S::GUS) roots after rhizobial inoculation, among them 268 genes were upregulated and 1122 genes were downregulated. Among downregulated genes, many known regulators of legume-rhizobia symbiosis were found. Genes upregulated in rhizobia-inoculated MtCLE35-overexpressing roots included ones associated with defense response and cellular redox status. Furthermore, stable transgenic plants overexpressing the MtCLE35 gene were obtained in this study, and gene expression qPCR analysis of selected differentially expressed genes in rhizobia-inoculated roots was performed in such plants. Collectively, our data suggest that overexpression of the MtCLE35 gene prevents the induction of nodulation program, which is accompanied by the expression of defense-related gene in MtCLE35-overexpressing rhizobia-inoculated M. truncatula roots. [ABSTRACT FROM AUTHOR]

The mitochondrial calcium uniporters (MCUs) are a family of calcium unidirectional transporters important for cytoplasmic Ca2+ signals. Though the MCU proteins in several plant species have been investigated, genome-wide analysis of MCUs in alfalfa is lacking. Here, via genome-wide analysis, a total of 5, 20, and 6 MCU genes were identified in three different alfalfa cultivars, namely Medicago truncatula Jemalong A17, Medicago sativa XinJiangDaYe, and M. sativa Zhongmu No. 1, respectively. They were further phylogenetically classified into three subfamilies. Most MCU genes have only one intron, and gene duplication events of MCU genes were observed within each alfalfa accession and between different accessions. All alfalfa MCU proteins contained a highly conserved MCU domain and 10 conserved motifs, featuring two transmembrane domains and a DI/VME motif. According to the tissue expression data of M. sativa Zhongmu No. 1, MsMCU6.2 was the most abundant transcript with the highest expression in the leaf, and MsMCU5 and MsMCU1.2 showed higher expression levels in the stem than other tissues. We analyzed the expression profiles of five MCU genes (MsMCU1.1/1.2/5/6.1/6.2) under salt, drought, and cold stresses via qRT-PCR assays. All five MCU genes were induced by drought stress, except MsMCU5, whose expression was up-regulated by salt stress, while cold stress slightly altered MsMCU expression. Nine potential interacting proteins and three miRNAs targeting MtMCUs were predicted. These results provide detailed knowledge of alfalfa MCU genes and suggest their potential functions in abiotic stress response. [ABSTRACT FROM AUTHOR]

Salt stress is a significant abiotic factor affecting the growth and development of alfalfa. Malondialdehyde (MDA) serves as a critical biomarker for assessing alfalfa's salt tolerance. Traditional methods for measuring MDA are often time-consuming and labor-intensive. Recent advances in remote sensing technology have made non-destructive estimation of metabolites feasible, positioning the accurate estimation of MDA content in alfalfa as a key focus in intelligent breeding. To address the challenge of detecting subtle changes in MDA content, this study developed a partial least squares regression (PLSR) model specifically for Medicago truncatula. This study utilized leaf reflectance hyperspectral data across the visible near-infrared–shortwave infrared (VIR-NIR-SWIR) spectrum, applying multi-order spectral transformation methods, including continuous wavelet transform (CWT), fractional differential (FD), and multi-granularity spectral segmentation (MGSS). Feature selection techniques, such as sequential forward selection (SFS), Least-Squares Boosting (LSBoost), and feature selection using neighborhood component analysis for regression (FSRNCA), were employed to enhance the efficiency of the MDA estimation. The findings revealed that the optimal PLSR model for MDA estimation was achieved by integrating CWT features across orders 1–30 with the SFS method. This model demonstrated robust estimation capabilities under varying salt stress conditions, significantly outperforming the original spectral data (R2 = 0.654, RMSE = 22.567 vs. R2 = 0.242, RMSE = 33.411). A comparative analysis of feature selection methods confirmed that SFS was the most effective for estimating MDA content in alfalfa. These results provide valuable insights and methodologies for MDA estimation and evaluating salt tolerance in alfalfa. [ABSTRACT FROM AUTHOR]

The imperative need to produce safe foodstuffs using environmentally sustainable practices has highlighted the incorporation of legumes in human and animal diets as an emerging nutritional staple. Since legumes comprise a family of plants known to display an extensive agricultural diversity with significant bioactivities, we report herein the exploitation outcome of the nutritional and bio-functional content of hay, derived from the post-harvest aerial parts of eight novel-improved Greek cultivars belonging to the following six taxa: Larthyrus sativus L., Medicago sativa L., Cicer arietinum L., Pisum sativum L., Vicia faba L., and Phaseolus vulgaris L. In specific, the phytochemical content of their hay extracts was screened against the presence of 37 fatty acids and 67 polyphenols using, respectively, GC-FID and LC-MS/MS instruments, while the determination of their total phenolic and tannin contents was achieved with spectrophotometric methods. In this respect, the presence of 26 different fatty acids was detected of which the acids linoleic, linolenic and palmitic were traced in all extracts in concentrations exceeding the 10 mg/g, while the oleic acid was determined as the major component of C. arietinum (77.58 mg/g), V. faba (36.11 mg/g), and L. sativus (12.89 mg/g) extracts. In addition, 30 different phenolics were traced as extracts' components with the most abundant the molecule of isoquercetin, which was present in six extracts and isoliquiritigenin, calycosin, and chlorogenic acid which were detected in five extracts. Finally, the antioxidant properties of the extracts were evaluated by performing both DPPH• and FRAP assays, which highlighted the V. faba extract as the most potent in both tests, followed by the extracts of P. sativum and P. vulgaris. Results herein are indicative of the significant advances achieved, for the improvement of investigated plant cultivars with respect to their utilization as nutritional crops, since these novel cultivars hays have been found to contain significant amounts of essential nutrients in the form of fatty acids along with bioactive ingredients in the form of polyphenols, while simultaneously they were determined as deprived of tannins that constitute essential anti-nutritional factors. [ABSTRACT FROM AUTHOR]

The emergence of commensalism and mutualism often derives from ancestral parasitism. However, in the case of rhizobium-legume interactions, bacterial strains displaying both pathogenic and nodulation features on a single host have not been described yet. Here, we isolated such a bacterium from Medicago nodules. On the same plant genotypes, the T4 strain can induce ineffective nodules in a highly competitive way and behave as a harsh parasite triggering plant death. The T4 strain presents this dual ability on multiple legume species of the Inverted Repeat-Lacking Clade, the output of the interaction relying on the developmental stage of the plant. Genomic and phenotypic clustering analysis show that T4 belongs to the nonsymbiotic Ensifer adhaerens group and clusters together with T173, another strain harboring this dual ability. In this work, we identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host. In plant-bacteria symbiosis, frontiers between mutualism and parasitism are often crossed. Here, the authors identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host. [ABSTRACT FROM AUTHOR]

HD-Zip proteins are plant-specific transcription factors known for their diverse functions in regulating plant growth, development, and responses to environmental stresses. Among the Medicago truncatula HD-Zip II genes, MtHB2 has been previously linked to abiotic stress responses. In this study, we conducted a functional characterization of MtHB2 in the regulation of root growth and development. Upon auxin stimulation, expression of MtHB2 was promptly up-regulated. Overexpression of MtHB2 in Arabidopsis thaliana led to reduced primary root growth and inhibited lateral root formation. Interestingly, the transgenic plants expressing MtHB2 exhibited differential responses to three types of auxins (IAA, NAA, and 2,4-D) in terms of root growth and development compared to the wild-type plants. Specifically, primary root growth was less affected, and lateral root formation was enhanced in the transgenic plants when exposed to auxins. This differential response suggests a potential role for MtHB2 in modulating auxin transport and accumulation, as evidenced by the reduced sensitivity of the transgenic plants to the auxin transport inhibitor NPA and lower expression levels of auxin-related reporters such as PIN-FORMED (PIN1)::PIN1-GFP, PIN3::PIN3-GFP, PIN7::PIN7-GFP, and DR5::GFP compared to wild-type plants. Additionally, microarray analysis of the root tissues revealed downregulation of several auxin-responsive genes in transgenic seedlings compared to wild-type plants. These findings collectively indicate that MtHB2 plays a critical regulatory role in root growth and development by modulating auxin accumulation and response in the roots. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1–3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN–NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation. NODULE INCEPTION directly binds the promoter of the nitrogen-fixing clade-specific gene NODULES WITH ACTIVATED DEFENSE1 to regulate rhizobial intracellular accommodation in nodules. [ABSTRACT FROM AUTHOR]

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2‐fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae‐gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae‐gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon–intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae‐gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae‐gain genetic innovation and sub‐functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae. [ABSTRACT FROM AUTHOR]

Forage legumes face simultaneous abiotic and biotic stresses, causing substantial yield losses. This study explores the combined impacts of drought and Phoma medicaginis infection on the growth and photosynthetic activity of Medicago truncatula contrasting lines TN6.18 and F83005.5. The tolerant TN6.18 line exhibits superior tolerance to combined drought and P. medicaginis infection, manifesting in minimal leaf area reduction, the highest number of healthy leaves, increased carotenoids content, a consistently high and stable photosynthesis rate, and enhanced performance of photosystems PSI and PSII. On the contrary, the sensitive F83005.5 line shows pronounced leaf chlorosis, particularly under drought stress, and decreased pigment levels under the combination of drought and P. medicaginis infection stresses (combined stress). Moreover, the drought-stressed F83005.5, experiences reduced hydration and photosynthetic performance, linked to diminished gas exchange and chlorophyll fluorescence parameters. Chlorophyll fluorescence revealed more severe PSI impairment than PSII under combined stress. In conclusion, understanding the Pm8 infection-drought interaction enhances insights into M. truncatula resistance mechanisms to the combination of these stresses. [ABSTRACT FROM AUTHOR]

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans -zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system. [ABSTRACT FROM AUTHOR]

The Arabidopsis SUPERMAN (SUP) gene and its orthologs in eudicots are crucial in regulating the number of reproductive floral organs. In Medicago truncatula, in addition to this function, a novel role in controlling meristem activity during compound inflorescence development was assigned to the SUP‐ortholog (MtSUP). These findings led us to investigate whether the role of SUP genes in inflorescence development was legume‐specific or could be extended to other eudicots. To assess that, we used Solanum lycopersicum as a model system with a cymose complex inflorescence and Arabidopsis thaliana as the best‐known example of simple inflorescence. We conducted a detailed comparative expression analysis of SlSUP and SUP from vegetative stages to flower transition. In addition, we performed an exhaustive phenotypic characterisation of two different slsup and sup mutants during the plant life cycle. Our findings reveal that SlSUP is required for precise regulation of the meristems that control shoot and inflorescence architecture in tomato. In contrast, in Arabidopsis, SUP performs no meristematic function, but we found a role of SUP in floral transition. Our findings suggest that the functional divergence of SUP‐like genes contributed to the modification of inflorescence architecture during angiosperm evolution. [ABSTRACT FROM AUTHOR]

Medicago truncatula is a key model plant for studying legume plants, particularly alfalfa (Medicago sativa), due to its well-defined genetic background. Plant-specific GASA (Gibberellic Acid Stimulated Arabidopsis) genes play various roles in plant growth and development, abiotic stress, and hormone responses. However, limited information is available on GASA research in Medicago. In this study, 26 MtGASAs were identified and analyzed for its structure, evolution, and expressions. Sequence alignments and phylogeny revealed that 26 MtGASAs containing conserved GASA domains were classified into three clades. The chromosomal locations and gene synteny revealed segmental and tandem repetition evolution. Analysis of cis-regulatory elements indicates that family members likely influence various hormone signaling pathways and stress-related mechanisms. Moreover, the RNA-seq and qRT-PCR analyses revealed that 26 MtGASAs were extensively involved in abiotic stresses and hormone responses. Notably, seven MtGASA genes (MtGASA1, 10, 12, 17, 23, 25 and 26) were all dramatically activated by NaCl and Mannitol treatments, and four MtGASAs (MtGASA7, 10, 23 and 24) were significant activated by GA3, PBZ, ABA, and MeJA treatments. Collectively, this study is the first to identify and describe GASA genes in Medicago on a genome-wide scale. The results establish a basis for functional characterization, showing that these proteins are essential in responding to various abiotic stresses and hormonal signals. [ABSTRACT FROM AUTHOR]

Nitrogen (N)-fixing symbiosis is critical to terrestrial ecosystems, yet possession of this trait is known for few plant species. Broader presence of the symbiosis is often indirectly determined by phylogenetic relatedness to taxa investigated via manipulative experiments. This data gap may ultimately underestimate phylogenetic, spatial, and temporal variation in N-fixing symbiosis. Still needed are simpler field or collections-based approaches for inferring symbiotic status. N-fixing plants differ from non-N-fixing plants in elemental and isotopic composition, but previous investigations have not tested predictive accuracy using such proxies. Here we develop a regional field study and demonstrate a simple classification model for fixer status using nitrogen and carbon content measurements, and stable isotope ratios (δ15N and δ13C), from field-collected leaves. We used mixed models and classification approaches to demonstrate that N-fixing phenotypes can be used to predict symbiotic status; the best model required all predictors and was 80–94% accurate. Predictions were robust to environmental context variation, but we identified significant variation due to native vs. non-native (exotic) status and phylogenetic affinity. Surprisingly, N content—not δ15N—was the strongest predictor, suggesting that future efforts combine elemental and isotopic information. These results are valuable for understudied taxa and ecosystems, potentially allowing higher-throughput field-based N-fixer assessments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Host range specificity is a prominent feature of the legume-rhizobial symbiosis. Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species that engage in root nodule symbiosis with legume plants of the Medicago genus, but certain Medicago species exhibit selectivity in their interactions with the two rhizobial species. We have identified a Medicago receptor-like kinase, which can discriminate between the two bacterial species, acting as a genetic barrier against infection by most S. medicae strains. Activation of this receptor-mediated nodulation restriction requires a bacterial gene that encodes a glycine-rich octapeptide repeat protein with distinct variants capable of distinguishing S. medicae from S. meliloti. This study sheds light on the coevolution of host plants and rhizobia, shaping symbiotic selectivity in their respective ecological niches. [ABSTRACT FROM AUTHOR]

Legumes are important grains and forages, providing high-quality proteins, vitamins, and micronutrients to humans and animals. Medicago truncatula is a close relative of alfalfa (Medicago sativa). Caffeic acid O-methyltransferase (COMT), a key gene that is identified to be essential for melatonin synthesis, plays a significant role in plant growth, development, and abiotic stress responses. However, a systematic study on the COMT gene family in M. truncatula has still not been reported. In this study, 63 MtCOMT genes were identified and categorized into three groups. Gene structure and conserved motif analyses revealed the relative conservation of closely clustered MtCOMTs within each group. Duplicated events in MtCOMT members were identified, and segmental duplication was the main mean. Cis-acting element prediction revealed the involvement of MtCOMTs in growth and development and response to light, stress, and plant hormones. RNA-seq data analysis showed that 57 MtCOMTs varied under salt and drought stresses. The RT-qPCR expression patterns showed that MtCOMT9, MtCOMT13, MtCOMT22, MtCOMT24, MtCOMT43, and MtCOMT46 were related to salt and drought responses in M. truncatula. Additionally, Arabidopsis thaliana overexpressing MtCOMT13 displayed superior plant growth phenotypes and enhanced tolerance to salt and drought stresses through higher photosynthetic parameters and activities of antioxidant enzymes, which indicated that MtCOMT13 played an important role in positively regulating plant salt and drought tolerance. These findings contribute to an improved understanding of MtCOMTs' roles in abiotic stress responses in M. truncatula, providing an important theoretical basis and genetic resource for legume species resistance breeding in the future. [ABSTRACT FROM AUTHOR]

Galactinol synthase (GolS) serves as a pivotal enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and assumes a crucial role in mediating plant responses to environmental stresses. However, a comprehensive exploration of the GolS gene family in the M. truncatula remains lacking. In this study, we identified five GolS genes (MtGolS) from the M. truncatula genome, and an analysis of evolution, domain, tissue expression, as well as their responses to various abiotic stresses and plant hormones was performed on these five genes. Comparative analysis revealed that the gene structure and domains of MtGolSs corresponded with those observed in GolS proteins in rice and Arabidopsis. Notably, the theoretical isoelectric point (pI) of MtGolS3 (8.31) was significantly surpassed that of its counterparts. And a segmental duplication event was found between MtGolS1 and MtGolS5. Furthermore, the expression patterns exhibited distinctive patterns for MtGolS3, characterized by predominant expression in flowers and shoot apical meristems, whereas MtGolS1, 2, 4, and 5 displayed heightened expression levels in seeds. Examination of promoters and expression profiles disclosed the responsiveness of the MtGolS genes to diverse abiotic stresses and phytohormone treatments. Specifically, MtGolS1, 2, 4, and 5 were significantly induced under drought, cold, and salt stress conditions. Additionally, MtGolS1, 3, 4, and 5 were upregulated by auxin (IAA) and gibberellin (GA), but inhibited by abscisic acid (ABA). Additionally, miRNA prediction analysis identified a total of 23 miRNA target sites within the five MtGolS genes, targeted by 17 distinct miRNAs. These findings furnish fundamental insights for further delving into the functional mechanisms of GolS genes in M. truncatula. [ABSTRACT FROM AUTHOR]

Summary: During arbuscular mycorrhizal (AM) symbiosis, plant innate immunity is modulated to a prime state to allow for fungal colonization. The underlying mechanisms remain to be further explored. In this study, two rice genes encoding LysM extracellular (LysMe) proteins were investigated.By obtaining OsLysMepro:GUS transgenic plants and generating oslysme1, oslysme2 and oslysme1oslysme2 mutants via CRISPR/Cas9 technique, OsLysMe genes were revealed to be specifically induced in the arbusculated cells and mutations in either gene caused significantly reduced root colonization rate by AM fungus Rhizophagus irregularis. Overexpression of OsLysMe1 or OsLysMe2 dramatically increased the colonization rates in rice and Medicago truncatula.The electrophoretic mobility shift assay and dual‐luciferase reporter assay supported that OsLysMe genes are regulated by OsWRI5a. Either OsLysMe1 or OsLysMe2 can efficiently rescue the impaired AM phenotype of the mtlysme2 mutant, supporting a conserved function of LysMe across monocotyledonous and dicotyledonous plants.The co‐localization of OsLysMe proteins with the apoplast marker SP‐OsRAmy3A implies their probable localization to the periarbuscular space (PAS) during symbiosis. Relative to the fungal biomass marker RiTEF, some defense‐related genes showed disproportionately high expression levels in the oslysme mutants. These data support that rice plants deploy two OsLysMe proteins to facilitate AM symbiosis, likely by diminishing plant defense responses. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. [ABSTRACT FROM AUTHOR]

Premise Methods Results Conclusions Legumes establish mutualistic interactions with pollinators and nitrogen (N)‐fixing bacteria that are critical for plant reproduction and ecosystem functioning. However, we know little about how N‐fixing bacteria and soil nutrient availability affect plant attractiveness to pollinators.In a two‐factorial greenhouse experiment to assess the impact of N‐fixing bacteria and soil types on floral traits and attractiveness to pollinators in Chamaecrista latistipula (Fabaceae), plants were inoculated with N‐fixing bacteria (NF+) or not (NF‐) and grown in N‐rich organic soil (+N organic soil) or N‐poor sand soil (‐N sand soil). We counted buds and flowers and measured plant size during the experiment. We also measured leaf, petal, and anther reflectance with a spectrophotometer and analyzed reflectance curves. Using the bee hexagon model, we estimated chromatic contrasts, a crucial visual cues for attracting bees that are nearby and more distant.NF+ plants in ‐N sand soil had a high floral display and color contrasts. On the other hand, NF‐ plants and/or plants in +N organic soil had severely reduced floral display and color contrasts, decreasing floral attractiveness to bee pollinators.Our findings indicate that the N‐fixing bacteria positively impact pollination, particularly when nutrients are limited. This study provides insights into the dynamics of plant–pollinator interactions and underscores the significant influence of root symbionts on key floral traits within tropical ecosystems. These results contribute to understanding the mechanisms governing mutualisms and their consequences for plant fitness and ecological dynamics. [ABSTRACT FROM AUTHOR]

Herbarium specimens are increasingly being used as sources of information to understand the ecology and evolution of plants and their associated microbes. Most studies have used specimens as a source of genetic material using culture‐independent approaches. We demonstrate that herbarium specimens can also be used to culture nodule‐associated bacteria, opening the possibility of using specimens to understand plant–microbe interactions at new spatiotemporal scales. We used historic and contemporary nodules of a common legume, Medicago lupulina, to create a culture collection. We were able to recover historic bacteria in 15 genera from three specimens (collected in 1950, 2004, and 2015). This work is the first of its kind to isolate historic bacteria from herbarium specimens. Future work should include inoculating plants with historic strains to see if they produce nodules and if they affect plant phenotype and fitness. Although we were unable to recover any Ensifer, the main symbiont of Medicago lupulina, we recovered some other potential nodulating species, as well as many putative growth‐promoting bacteria. [ABSTRACT FROM AUTHOR]

This article explores the role of a protein called VAPYRIN (VPY) in the process of rhizobial infection in legume plants. Rhizobial infection is necessary for legumes to fix nitrogen, and the study focuses on the specific amino acid Asp394 in VPY. The research found that Asp394 is crucial for the interaction between VPY and another protein called LIN, as well as for the localization and function of VPY during infection. The study suggests that Asp394 may play a significant role in the interaction between VPY and other components of the infectosome complex, but further research is needed to fully understand its function in plant-microbe interactions. [Extracted from the article]

The ecologically and economically vital symbiosis between nitrogen-fixing rhizobia and leguminous plants is often thought of as a bi-partite interaction, yet studies increasingly show the prevalence of non-rhizobial endophytes (NREs) that occupy nodules alongside rhizobia. Yet, what impact these NREs have on plant or rhizobium fitness remains unclear. Here, we investigated four NRE strains found to naturally co-occupy nodules of the legume Medicago truncatula alongside Sinorhizobium meliloti in native soils. Our objectives were to (1) examine the direct and indirect effects of NREs on M. truncatula and S. meliloti fitness, and (2) determine whether NREs can re-colonize root and nodule tissues upon reinoculation. We identified one NRE strain (522) as a novel Paenibacillus species, another strain (717A) as a novel Bacillus species, and the other two (702A and 733B) as novel Pseudomonas species. Additionally, we found that two NREs (Bacillus 717A and Pseudomonas 733B) reduced the fitness benefits obtained from symbiosis for both partners, while the other two (522, 702A) had little effect. Lastly, we found that NREs were able to co-infect host tissues alongside S. meliloti. This study demonstrates that variation of NREs present in natural populations must be considered to better understand legume–rhizobium dynamics in soil communities. [ABSTRACT FROM AUTHOR]

Biochars produced from different feedstocks and at different pyrolysis temperatures may have various chemical and physical properties, affecting their potential use as alternative microbial carrier materials. In this study, biochars were produced from pine wood and oak feedstocks at various temperatures (400°C, 500°C, 600°C, 700°C and 800°C), characterized, and assessed for their potential as carriers for Bradyrhizobium japonicum (CB1809) strain. The biochars were then stored at two different storage temperatures (28°C and 38°C) for up to 90 days. Furthermore, the study also explored the role of potentially ideal carriers as inoculants in the growth of Glycine max L. (soybean) under different moisture levels i.e., 55% water holding capacity (WHC) (D0), 30% WHC (D1) and, 15% WHC (D2) using a mixture of 50% garden soil and 50% sand. The results were compared to a control group (without inoculants) and a peat inoculant. Among all the materials derived from pine wood and oak, pine wood biochar pyrolyzed at 400℃ (P-BC400) exhibited the highest CFU count, with values of 10.34 and 9.74 Log 10 CFU g− 1 after 90 days of storage at 28℃ and 38℃, respectively. This was notably higher compared to other biochars and peat carriers. Significant (p < 0.05) increases in plant properties: shoot and root dry biomass (174% and 367%), shoot and root length (89% and 85%), number of leaves (71%), membrane stability index (27%), relative water content (26%), and total chlorophyll (140%) were observed in plants treated with P-BC400 carrier inoculant compared to the control at D2; however, lower enrichment of δ13C (37%) and δ15N (108%) with highest number of root nodules (8.3 ± 1.26) and nitrogenase activity (0.869 ± 0.04) were observed under D2, as evident through PCA analysis, showing more nitrogen (N) fixation and photosynthetic activity. Overall, this experiment concluded that biochar pyrolyzed at lower temperatures, especially P-BC400, was the most suitable candidate for rhizobial inoculum and promoted soybean growth. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Dynamic shift in methylation happens during the somatic embryogenesis process in Medicago truncatula. [ABSTRACT FROM AUTHOR]

Alfalfa (Medicago sativa L.) is a vitally important perennial fodder legume worldwide. Given their particular traits, alfalfa crop wild relatives (CWRs) could be used to develop cultivars that can tolerate extreme environmental and climatic conditions. Until now, researchers have overlooked the composition and structure of bacterial communities in the root zone of alfalfa and its relevant CWRs and their influence on forage performance under actual field conditions. In this study, high-throughput sequencing of 16S rRNA analysis was performed to investigate the diversity and assemblies of bacterial communities in the bulk soil and in the root zone of individual field-grown Medicago plants arranged in a honeycomb selection design. The plants used in this study were M. sativa × M. arborea hybrids (Genotypes 6 and 8), the closely-related M. sativa nothosubsp. varia (Martyn) Arcang. (Genotype 13), and M. sativa ssp. sativa (Genotype 20). The bacterial communities in the root-zone samples and the assemblies in the bulk soil differed significantly. Genotype 13 was found to have distinct bacterial assemblies from the other genotypes while exhibiting the lowest forage productivity. These findings suggest that plant productivity may influence the composition of bacterial communities in the root zone. Biomarker analysis conducted using linear discriminant analysis (LDA) revealed that only members of the Rhizobiales order were enriched in the M. sativa nothosubsp. varia root zone whereas taxa belonging to Sphingomonas and various Bacteriodota were enriched in the other genotypes. Of the shared taxa identified in the root zone of the Medicago lines, the abundance of specific taxa, namely, Flavisolibacter, Stenotrophomonas, and Sphingomonas, were positively associated with forage yield. This pioneering study, in which the root zones of individual Medicago plants under actual field conditions were examined, offers evidence of differences in the bacterial composition of alfalfa genotypes with varying genetic backgrounds. Its findings indicate that particular bacterial taxa may favorably influence plant performance. This study covered the first six months of crop establishment and paves the way for further investigations to advance understanding of how shifts in bacterial assemblies in alfalfa roots affect plant performance over time. [ABSTRACT FROM AUTHOR]

The mechanisms of how plant-beneficial rhizospheric fungi interact with the soil microbial community to promote plant growth by facilitating their phosphorus acquisition are poorly understood. This work supported that a Mucoromycotina fungus, Gongronella sp. w5 (w5), could promote phosphorus uptake of Medicago truncatula by increasing the available phosphorus (P) in the soil. The abundance of phosphate-solubilizing bacteria (PSB) and the activity of alkaline phosphatase (ALP) in alfalfa rhizosphere soil increased after w5 inoculation. Further analysis showed that w5 donated a portion of ALP activity and also stimulated the PSB to secrete ALP during plant-w5-PSB interaction to help release more available P in the rhizosphere of M. truncatula. Unlike most plant-beneficial rhizospheric fungi that mainly acquire hexoses from plants, w5 gained sucrose directly from the host plant and then recruited PSB to aid P acquisition by hydrolyzing sucrose and releasing mainly fructose to induce PSB to secrete ALP. [ABSTRACT FROM AUTHOR]

Introduction: The production of highly vigorous seeds with high longevity is an important lever to increase crop production efficiency, but its acquisition during seed maturation is strongly influenced by the growth environment. Methods: An association rule learning approach discovered MtABI4, a known longevity regulator, as a gene with transcript levels associated with the environmentally-induced change in longevity. To understand the environmental sensitivity of MtABI4 transcription, Yeast One-Hybrid identified a class I BASIC PENTACYSTEINE (MtBPC1) transcription factor as a putative upstream regulator. Its role in the regulation of MtABI4 was further characterized. Results and Discussion: Overexpression of MtBPC1 led to a modulation of MtABI4 transcripts and its downstream targets. We show that MtBPC1 represses MtABI4 transcription at the early stage of seed development through binding in the CT-rich motif in its promoter region. To achieve this, MtBPC1 interacts with SWINGER, a sub-unit of the PRC2 complex, and Sin3-associated peptide 18, a sub-unit of the Sin3-like deacetylation complex. Consistent with this, developmental and heat stress-induced changes in MtABI4 transcript levels correlated with H3K27me3 and H3ac enrichment in the MtABI4 promoter. Our finding reveals the importance of the combination of histone methylation and histone de-acetylation to silence MtABI4 at the early stage of seed development and during heat stress. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is one of the most destructive pathogens that threaten rice production around the world. Previous studies mainly focus on pathogenic mechanism of M. oryzae during infection on rice at leaf stage. However, the pathogenic mechanism of M. oryzae infection on panicle tissue is not well understood. In the present study, we performed RNA sequencing (RNA-seq) to study gene expression patterns of M. oryzae during infection at leaf stage and at panicle stage, respectively. The differentially expressed genes (DEGs) of M. oryzae in the infected leaf and panicle tissues were analyzed. Gene ontology (GO) enrichment analysis of DEGs revealed that M. oryzae genes involved in the biological processes were different at leaf and panicle stages. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs indicates that genes related to individual and important pathways may function at different infection stages. In particular, CAZymes carbohydrate esterases (CEs), carbohydrate-binding modules (CBMs), and glycoside hydrolases (GHs) may play important roles during M. oryzae infection on rice leaves, while glycosyltransferases (GTs) and GHs may play important roles during infection at rice panicle stage. Further analysis of effectors (BAS3, BAS113, BAS162, MoCDIP4, and MoHEG13) and their homologous genes suggest that they are involved in host defense suppression. Our findings provide insights into understanding the infection mechanisms of M. oryzae for rice leaf blast and panicle blast disease. [ABSTRACT FROM AUTHOR]

Lespedeza davurica (Laxm.) is a leguminous plant with significant ecological benefits, but its embryonic development mechanism remains unclear. We investigated the flower bud differentiation, megaspore and microspore formation, gametophyte development, and embryo and endosperm development in L. davurica. Our aim was to elucidate the relationship between the external morphology and internal development processes of male and female floral organs during growth, as well as the reproductive factors influencing fruiting. The results indicated that although the pistil develops later than the stamen during flower bud differentiation, both organs mature synchronously before flowering. L. davurica pollen exhibits three germination grooves, a reticulate outer wall, and papillary structures on the anther surface. In vivo pollination experiments revealed abnormal spiral growth of L. davurica pollen tubes within the style and the occurrence of callus plugs, which may reduce the seed setting rate. The anther wall development follows the dicotyledonous type, with tetrads formed through microspore meiosis exhibiting both left–right symmetry and tetrahedral arrangements. L. davurica has a single ovule, and the embryo sac develops in the monosporic polygonum type. After dormancy, the zygote undergoes multiple divisions, progressing through spherical, heart-shaped, and torpedo-shaped embryo stages, culminating in a mature embryo. A mature seed comprises cotyledons, hypocotyl, embryo, radicle, and seed coat. Phylogenetic tree analysis reveals a close genetic relationship between L. davurica and other leguminous plants from the genera Lespedeza and Medicago. This study provides valuable insights into the regulation of flowering and hybrid breeding in leguminous plants and offers a new perspective on the development of floral organs and seed setting rates. [ABSTRACT FROM AUTHOR]

Given the escalating impact of climate change on agriculture and food security, gaining insights into the evolutionary dynamics of climatic adaptation and uncovering climate-adapted variation can empower the breeding of climate-resilient crops to face future climate change. Alfalfa (Medicago sativa subsp. sativa), the queen of forages, shows remarkable adaptability across diverse global environments, making it an excellent model for investigating species responses to climate change. In this study, we performed population genomic analyses using genome resequencing data from 702 accessions of 24 Medicago species to unravel alfalfa's climatic adaptation and genetic susceptibility to future climate change. We found that interspecific genetic exchange has contributed to the gene pool of alfalfa, particularly enriching defense and stress-response genes. Intersubspecific introgression between M. sativa subsp. falcata (subsp. falcata) and alfalfa not only aids alfalfa's climatic adaptation but also introduces genetic burden. A total of 1671 genes were associated with climatic adaptation, and 5.7% of them were introgressions from subsp. falcata. By integrating climate-associated variants and climate data, we identified populations that are vulnerable to future climate change, particularly in higher latitudes of the Northern Hemisphere. These findings serve as a clarion call for targeted conservation initiatives and breeding efforts. We also identified pre-adaptive populations that demonstrate heightened resilience to climate fluctuations, illuminating a pathway for future breeding strategies. Collectively, this study enhances our understanding about the local adaptation mechanisms of alfalfa and facilitates the breeding of climate-resilient alfalfa cultivars, contributing to effective agricultural strategies for facing future climate change. Population genomic analyses are performed in thit study to unravel alfalfa's climatic adaptation and vulnerability to future climate change. It is found that intraspecific gene flow and interspecific introgression have introduced genes that facilitate adaptation to current and future climates. A total of 1671 candidate genes are identified as being associated with climatic adaptation, providing potential targets for the breeding of climate-resilient cultivars to cope with future climate change. [ABSTRACT FROM AUTHOR]

Summary: Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula.We biochemically characterized five flavonoid‐O‐methyltransferases (OMTs) and a lux‐based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids.We found that chalcone‐OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4′‐dihydroxy‐2′‐methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2.We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range. [ABSTRACT FROM AUTHOR]

This article explores the recruitment of arbuscular mycorrhizal (AM) conserved genes for ectomycorrhizal (ECM) symbiosis. ECM symbiosis, which is a more recent form of symbiosis than AM symbiosis, involves the formation of a mantle around the root and an intercellular Hartig net. The study found that ECM host species from the Fagales order maintained the majority of AM-conserved genes, unlike other non-AM hosts. Transcriptome analysis revealed that several of these maintained genes were induced in ECM symbiosis, suggesting that ECM formation recruits AM-derived mechanisms. The study focuses on ECM-only hosts, which are plant species that exclusively engage in ECM symbiosis and have lost the ability to form AM symbiosis. The researchers discovered that ECM-only hosts maintain 64% of the AM-conserved orthologous groups (OGs), highlighting the importance of these genes in ECM symbiosis. They also identified specific genes, such as RAM2, involved in lipid biosynthesis and arbuscule formation in ECM roots. The study suggests that the preservation of AM-conserved genes in ECM-only hosts supports their role in ECM symbiosis. [Extracted from the article]

Soybean [Glycine max (L.) Merr.] is one of the most important and oldest crops. Due to its ability to form symbiotic interactions with nitrogen-fixing bacteria, it is a valuable source of nitrogen for agriculture and proteins for humans and livestock. In Europe, for instance, in Poland, the soybean cultivation area is still not large but is gradually increasing due to climate change. The lack of indigenous soybean microsymbionts in Polish soils forces the application of commercial strains to establish effective symbioses. Fast and reliable identification methods are needed to study the persistence, competitiveness, and dispersal of bradyrhizobia introduced as inocula. Our study aimed to apply real-time PCR coupled with high-resolution melting curve (HRM) analysis to detect and differentiate bacterial strains occupying soybean nodules. HRM-PCR was performed on crude extracts from nodules using primers specific for recA, a highly conserved nonsymbiotic gene. By comparing them with the reference strains, we were able to identify and assign Bradyrhiobium strains that had been introduced into field locations in Poland. In conclusion, HRM analysis was proven to be a fast and accurate method for identifying soybean microsymbionts and might be successfully used for identifying other legume-nodulating bacteria. [ABSTRACT FROM AUTHOR]

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses. [ABSTRACT FROM AUTHOR]

Considering the medicinal and conservational significance of Saraca asoca, the present study employed three different geographical ranges for building ecological niche models. The vifstep procedure detected multicollinearity among 10 out of 19 predictor variables. The selected subset included mean diurnal range, isothermality, mean temperature of wettest quarter, mean temperature of driest quarter, annual precipitation, precipitation of driest month, precipitation seasonality, precipitation of warmest quarter, and precipitation of coldest quarter. The performances of machine learning and regression approaches were compared. Machine learning algorithm RF outweighed all other algorithms in performance. Following RF, model algorithms viz., Maxent, BRT, GLM, FDA, and Bioclim performed better in the declining order. Machine learning algorithms performed better than regression and profile-based approaches. The weighted average of True skill statistic was used to develop ensemble models. Potential habitats in native and introduced ranges in present and future conditions were identified. Introduction potential in unintroduced areas where herbal medicines were in greater use was also assessed. With rise in emissions, range of S. asoca may prefer an eastward expansion in native range and northward expansion in Andaman Nicobar Islands. If S. asoca is planted in recommended potential ranges in African and Latin American continents, eastward expansion in West Africa and westward expansion in Latin America may occur if temperature rises. The present study could develop a robust evidence-based hypothesis for ecologists, conservationists, herbal medicine manufactures, government agencies, and forest departments at national/international level to establish plantations for growing S. asoca. [ABSTRACT FROM AUTHOR]

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

Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock‐out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation. [ABSTRACT FROM AUTHOR]

Cold stress severely restricts plant development, causing significant agricultural losses. We found a critical transcription factor network in Medicago ruthenica was involved in plant adaptation to low‐temperature. APETALA2/ethylene responsive factor (AP2/ERF) transcription factor MrERF039 was transcriptionally induced by cold stress in M. ruthenica. Overexpression of MrERF039 significantly increased the glucose and maltose content, thereby improving the tolerance of M. ruthenica. MrERF039 could bind to the DRE cis‐acting element in the MrCAS15A promoter. Additionally, the methyl group of the 14th amino acid in MrERF039 was required for binding. Transcriptome analysis showed that MrERF039 acted as a sugar molecular switch, regulating numerous sugar transporters and sugar metabolism‐related genes. In addition, we found that MrERF039 could directly regulate β‐amylase gene, UDP glycosyltransferase gene, and C2H2 zinc finger protein gene expression. In conclusion, these findings suggest that high expression of MrERF039 can significantly improve the cold tolerance of M. ruthenica root tissues during cold acclimation. Our results provide a new theoretical basis and candidate genes for breeding new legume forage varieties with high resistance. Summary statement: MrERF039 plays a positive role in the cold response of Medicago ruthenica by directly regulating sugar‐related genes, including β‐amylase through direct binding to DRE cis‐acting elements, increasing glucose and maltose content, and consequently improving the cold tolerance of Medicago ruthenica. [ABSTRACT FROM AUTHOR]

Leaf movement is a manifestation of plant response to the changing internal and external environment, aiming to optimize plant growth and development. Leaf movement is usually driven by a specialized motor organ, the pulvinus, and this movement is associated with different changes in volume and expansion on the two sides of the pulvinus. Blue light, auxin, GA, H+-ATPase, K+, Cl−, Ca2+, actin, and aquaporin collectively influence the changes in water flux in the tissue of the extensor and flexor of the pulvinus to establish a turgor pressure difference, thereby controlling leaf movement. However, how these factors regulate the multicellular motility of the pulvinus tissues in a species remains obscure. In addition, model plants such as Medicago truncatula, Mimosa pudica, and Samanea saman have been used to study pulvinus-driven leaf movement, showing a similarity in their pulvinus movement mechanisms. In this review, we summarize past research findings from the three model plants, and using Medicago truncatula as an example, suggest that genes regulating pulvinus movement are also involved in regulating plant growth and development. We also propose a model in which the variation of ion flux and water flux are critical steps to pulvinus movement and highlight questions for future research. [ABSTRACT FROM AUTHOR]

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation. PINNATE-LIKE PENTAFOLIATA2, a boundary-expressed transcription factor, controls compound leaf morphogenesis by directly repressing the activity of a leaflet initiation regulator in Medicago truncatula. [ABSTRACT FROM AUTHOR]

Seed priming is routinely applied to improve germination rates and seedling establishment, but the decrease in longevity observed in primed seeds constitutes a major drawback that compromises long-term storability. The optimization of priming protocols able to preserve primed seeds from aging processes represents a promising route to expand the scope of seed priming. The present work explores this possibility in the model legume Medicago truncatula by testing the effectiveness of quercetin- and rutin-supplemented seed priming at improving the response to subsequent artificial aging. In comparison with a non-supplemented hydropriming protocol, supplementation with quercetin or rutin was able to mitigate the effects of post-priming aging by increasing germination percentage and speed, improving seed viability and seedling phenotype, with consistent correlations with a decrease in the levels of reactive oxygen species and an increase in antioxidant potential. The results suggest that quercetin and rutin can reduce the effects of post-priming aging by improving the seed antioxidant profiles. The present work provides novel information to explore the physiological changes associated with seed priming and aging, with possible outcomes for the development of tailored vigorization protocols able to overcome the storability constrains associated with post-priming aging processes. [ABSTRACT FROM AUTHOR]

A comparative metabolomic study of three varieties of alfalfa (Medicago varia Mart.) was performed via extraction with supercritical carbon dioxide modified with ethanol (EtOH) and the detection of bioactive compounds via tandem mass spectrometry. Several experimental conditions were investigated in the pressure range of 50–250 bar, with ethanol used as a co-solvent in an amount of 1% of the total volume in the liquid phase at a temperature in the range of 31–70 °C. The most effective extraction conditions were as follows: a pressure of 250 Bar and a temperature of 60 °C for M. varia. M. varia contains various phenolic compounds and sulfated polyphenols with valuable biological activity. Tandem mass spectrometry (HPLC-ESI–ion trap) was applied to detect the target analytes. A total of 103 bioactive compounds (59 polyphenols and 44 compounds belonging to other chemical groups) were tentatively identified in extracts from aerial parts of alfalfa. For the first time, twenty-one chemical constituents from the polyphenol group (flavones: Formononetin, Chrysoeriol, Cirsimaritin, Cirsiliol, Cirsilineol, tricin-O-hexoside, Apigenin C-glucose C-deoxyhexoside, Apigenin 7-O-diglucuronide, 2′-Hydroxygenistein 4′,7-O-diglucoside, etc.) and six from other chemical groups (saponins: Soyasaponin II, Soyasaponin gamma g, Soyasaponin I, Soyasaponin Bd, Soyaysaponin beta g, etc.) were identified in the aerial parts of M. varia. [ABSTRACT FROM AUTHOR]

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and speciesrepresentative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species. [ABSTRACT FROM AUTHOR]

Chickpea, being an important grain legume crop, is often confronted with the adverse effects of high temperatures at the reproductive stage of crop growth, drastically affecting yield and overall productivity. The current study deals with an extensive evaluation of chickpea genotypes, focusing on the traits associated with yield and their response to heat stress. Notably, we observed significant variations for these traits under both normal and high-temperature conditions, forming a robust basis for genetic research and breeding initiatives. Furthermore, the study revealed that yield-related traits exhibited high heritability, suggesting their potential suitability for marker-assisted selection. We carried out singlenucleotide polymorphism (SNP) genotyping using the genotyping-bysequencing (GBS) method for a genome-wide association study (GWAS). Overall, 27 marker--trait associations (MTAs) linked to yield-related traits, among which we identified five common MTAs displaying pleiotropic effects after applying a stringent Bonferroni-corrected p-value threshold of <0.05 [-log10(p) > 4.95] using the BLINK (Bayesian-information and linkagedisequilibrium iteratively nested keyway) model. Through an in-depth in silico analysis of these markers against the CDC Frontier v1 reference genome, we discovered that the majority of the SNPs were located at or in proximity to genecoding regions. We further explored candidate genes situated near these MTAs, shedding light on the molecular mechanisms governing heat stress tolerance and yield enhancement in chickpeas such as indole-3-acetic acid--amido synthetase GH3.1 with GH3 auxin-responsive promoter and pentatricopeptide repeat-containing protein, etc. The harvest index (HI) trait was associated with marker Ca3:37444451 encoding aspartic proteinase ortholog sequence of Oryza sativa subsp. japonica and Medicago truncatula, which is known for contributing to heat stress tolerance. These identified MTAs and associated candidate genes may serve as valuable assets for breeding programs dedicated to tailoring chickpea varieties resilient to heat stress and climate change. [ABSTRACT FROM AUTHOR]

Due to rapidly emerging resistance to single‐site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation‐tolerant MtDef4 variant was generated that bound to β‐glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation‐tolerant variant as a peptide‐based fungicide. [ABSTRACT FROM AUTHOR]

Meloidogyne hapla is a serious pest of many cultivated plants. In response to the economic significance of the species, efforts are being made to develop a new method to reduce its harmful effects on crops. The aim of the study was to determine the effect of diffusates from seeds of selected species of legumes on the motility of second-stage juveniles and to evaluate the effect of meal from seeds of selected species of legume on the capacity to infect the roots of tomato plants by the J2 stage. The experiment examined the effect of diffusates on the motility of the J2 stage performed in Petri dishes, at temperatures of 10 °C, 17 °C and 21 °C. The evaluation of the J2 stage infectivity was estimated in a pot experiment performed under controlled conditions of 20 ± 1 °C. The pots were filled with sterile substrate mixed with meal from the seeds of selected plants at 1%, 5% and 10% of the substrate weight. The studies carried out in the Petri dishes showed varying effects of the seed diffusates from selected legume plants on the motility of the J2 stage of Meloidogyne hapla. J2 were found to lose their motility within 24 h after immersion in water containing seed diffusates from Melilotus albus, Trifolium pratense T. repens, in the temperature ranges investigated (10 °C, 17 °C and 21 °C). However, in a mixture of seed diffusates and soil filtrate from the root zone of tomato plants, the absence of motility in the second-stage juveniles was observed after 24 h at 17 °C and 21 °C, with seed diffusates from Lotus corniculatus, Medicago sativa, Medicago × varia, Melilotus officinalis, as well as Onobrychis viciifolia, Ornithopus sativus, Vicia sativa, used in the mixture. Galega officinalis Risa (GoR) seed diffusates were found to have an inhibiting effect on the motility of the J2 stage of M. hapla 24 h following the immersion of the J2 stage in the solution of the soil filtrate containing tomato root diffusates, at 21 °C. The J2 stage were not rendered immotile in all the experiment set-ups involving the seeds of V. faba, Lupinus spp., likewise in the control set-ups. In the pots studied, a significant effect of the addition of legume seed meal on the development of M. hapla nematodes and tomato plants was found. The introduction of Lotus corniculatus, Onobrychis viciifolia and Vicia sativa seed meal into the substrate in the proportion of 1%, 5% and 10% resulted in the inhibition of the J2 stage penetration into the roots of tomato plants at temperatures of 17 °C and 21 °C. With the admixture of the M. sativa and T. repens seed meal, within the temperature range investigated, no nematode infection was observed in the roots, regardless of the seed meal content in the substrate. As regards to the fresh weight, tomato plants grown in a substrate containing 1% and 5% of the V. sativa cv. Jaga seed meal were characterised by significantly higher plant weight values as compared to those grown in the control set-up. The obtained results imply that is advisable to expand the scope of research to include other economically important crops damaged by the northern root-knot nematode. [ABSTRACT FROM AUTHOR]

Especially within the light of the current loss of biodiversity, we want our students to gain a better understanding of the issues at stake in order to take action and support plants and their pollinators. Many educational approaches focus on honeybees, disregarding the vast diversity in the context of pollination systems and the complex (co-)adaptations on both plants and pollinators that evolved over time. This article tries to remedy this by providing scientific background and two practical teaching approaches including legumes (Fabaceae s. s., Faboideae) as one of the largest plant families worldwide, containing many well-known crop species. Legume flowers show specializations regarding pollination and are therefore excellent examples to teach not only about pollination and adaptations, but also conservation. The main elements of both teaching approaches are functional flower models. The first approach was developed for learners with little background knowledge on pollination, whereas the second approach is more elaborate. [ABSTRACT FROM AUTHOR]