Three separate pathways in Rhizobium leguminosarum maintain phosphatidylcholine biosynthesis, which is required for symbiotic nitrogen fixation with clover.

Phosphatidylcholine (PC) is critical for the nitrogen-fixing symbiosis between rhizobia and legumes. We characterized three PC biosynthesis pathways in Rhizobium leguminosarum and evaluated their impact on nitrogen fixation in clover nodules. In the presence of choline, a PC synthase catalyzes the condensation of cytidine diphosphate-diacylglycerol with choline to produce PC. In the presence of lyso-PC, acyltransferases acylate this mono-acylated phospholipid to PC. The third pathway relies on phospholipid N-methyltransferases (Pmts), which sequentially methylate phosphatidylethanolamine (PE) through three rounds of methylation, yielding PC via the intermediates monomethyl-PE and dimethyl-PE. In R. leguminosarum, at least three Pmts participate in this methylation cascade. To elucidate the functions of these enzymes, we recombinantly produced and biochemically characterized them. We moved on to determine the phospholipid profiles of R. leguminosarum mutant strains harboring single and combinatorial deletions of PC biosynthesis genes. The cumulative results show that PC production occurs through the combined action of multiple enzymes, each with distinct substrate and product specificities. The methylation pathway emerges as the dominant PC biosynthesis route, and we pinpoint PmtS2, which catalyzes all three methylation steps, as the enzyme responsible for providing adequate PC amounts for a functional nitrogen-fixing symbiosis with clover. IMPORTANCE: Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.

Adaptation of Rhizobium leguminosarum sv. trifolii strains to low temperature stress in both free-living stage and during symbiosis with clover.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. This process occurs within special new structures, called nodules, formed mainly on legume roots. Soil bacteria, commonly known as rhizobia, fix atmospheric dinitrogen, converting it into a form that can be assimilated by plants. Various environmental factors, including a low temperature, have an impact on the symbiotic efficiency. Nevertheless, the effect of temperature on the phenotypic and symbiotic traits of rhizobia has not been determined in detail to date. Therefore, in this study, the influence of temperature on different cell surface and symbiotic properties of rhizobia was estimated. In total, 31 Rhizobium leguminosarum sv. trifolii strains isolated from root nodules of red clover plants growing in the subpolar and temperate climate regions, which essentially differ in year and day temperature profiles, were chosen for this analysis. Our results showed that temperature has a significant effect on several surface properties of rhizobial cells, such as hydrophobicity, aggregation, and motility. Low temperature also stimulated EPS synthesis and biofilm formation in R. leguminosarum sv. trifolii. This extracellular polysaccharide is known to play an important protective role against different environmental stresses. The strains produced large amounts of EPS under tested temperature conditions that facilitated adherence of rhizobial cells to different surfaces. The high adaptability of these strains to cold stress was also confirmed during symbiosis. Irrespective of their climatic origin, the strains proved to be highly effective in attachment to legume roots and were efficient microsymbionts of clover plants. However, some diversity in the response to low temperature stress was found among the strains. Among them, M16 and R137 proved to be highly competitive and efficient in nodule occupancy and biomass production; thus, they can be potential yield-enhancing inoculants of legumes.

Comparative Chloroplast Genomes Analysis Provided Adaptive Evolution Insights in Medicago ruthenica.

A perennial leguminous forage, Medicago ruthenica has outstanding tolerance to abiotic stresses. The genome of Medicago ruthenica is large and has a complex genetic background, making it challenging to accurately determine genetic information. However, the chloroplast genome is widely used for researching issues related to evolution, genetic diversity, and other studies. To better understand its chloroplast characteristics and adaptive evolution, chloroplast genomes of 61 Medicago ruthenica were assembled (including 16 cultivated Medicago ruthenica germplasm and 45 wild Medicago ruthenica germplasm). These were used to construct the pan-chloroplast genome of Medicago ruthenica, and the chloroplast genomes of cultivated and wild Medicago ruthenica were compared and analyzed. Phylogenetic and haplotype analyses revealed two main clades of 61 Medicago ruthenica germplasm chloroplast genomes, distributed in eastern and western regions. Meanwhile, based on chloroplast variation information, 61 Medicago ruthenica germplasm can be divided into three genetic groups. Unlike the phylogenetic tree constructed from the chloroplast genome, a new intermediate group has been identified, mainly consisting of samples from the eastern region of Inner Mongolia, Shanxi Province, and Hebei Province. Transcriptomic analysis showed that 29 genes were upregulated and three genes were downregulated. The analysis of these genes mainly focuses on enhancing plant resilience and adapting adversity by stabilizing the photosystem structure and promoting protein synthesis. Additionally, in the analysis of adaptive evolution, the accD, clpP and ycf1 genes showed higher average Ka/Ks ratios and exhibited significant nucleotide diversity, indicating that these genes are strongly positively selected. The editing efficiency of the ycf1 and clpP genes significantly increases under abiotic stress, which may positively contribute to plant adaptation to the environment. In conclusion, the construction and comparative analysis of the complete chloroplast genomes of 61 Medicago ruthenica germplasm from different regions not only revealed new insights into the genetic variation and phylogenetic relationships of Medicago ruthenica germplasm, but also highlighted the importance of chloroplast transcriptome analysis in elucidating the model of chloroplast responses to abiotic stress. These provide valuable information for further research on the adaptive evolution of Medicago ruthenica.

Analyzing Medicago spp. seed morphology using GWAS and machine learning.

Alfalfa is widely recognized as an important forage crop. To understand the morphological characteristics and genetic basis of seed morphology in alfalfa, we screened 318 Medicago spp., including 244 Medicago sativa subsp. sativa (alfalfa) and 23 other Medicago spp., for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness & red-green-blue (RGB) intensities. The results revealed phenotypic diversity and correlations among the tested accessions. Based on the phenotypic data of M. sativa subsp. sativa, a genome-wide association study (GWAS) was conducted using single nucleotide polymorphisms (SNPs) called against the Medicago truncatula genome. Genes in proximity to associated markers were detected, including CPR1, MON1, a PPR protein, and Wun1(threshold of 1E-04). Machine learning models were utilized to validate GWAS, and identify additional marker-trait associations for potentially complex traits. Marker S7_33375673, upstream of Wun1, was the most important predictor variable for red color intensity and highly important for brightness. Fifty-two markers were identified in coding regions. Along with strong correlations observed between seed morphology traits, these genes will facilitate the process of understanding the genetic basis of seed morphology in Medicago spp.

Species-specific microsymbiont discrimination mediated by a Medicago receptor kinase.

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.

Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses.

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.

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

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

Over-expression of Medicago Acyl-CoA-binding 2 genes enhance salt and drought tolerance in Arabidopsis.

Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and they function in lipid metabolism, membrane biosynthesis, cellular signaling, stress response, disease resistance, and other biological activities in plants. However, the roles of ACBP family members in Medicago remain unclear. In this study, a total of eight ACBP genes were identified in the genome of Medicago truncatula and Medicago sativa, and they were clustered into four sub-families (Class I-IV). Many cis-acting elements related to abiotic response were identified in the promoter region of these ACBP genes, in particular light-responsive elements. These ACBP genes exhibited distinct expression pattern in various tissues, and the expression level of MtACBP1/MsACBP1 and MtACBP2/MsACBP2 gene pairs were significantly increased under NaCl treatment. Subcellular localization analysis showed that MtACBP1/MsACBP1 and MtACBP2/MsACBP2 were localized in the endoplasmic reticulum of tobacco epidermal cells. Arabidopsis seedlings over-expressing MtACBP2/MsACBP2 displayed increased root length than the wild type under short light, Cu2+, ABA, PEG, and NaCl treatments. Over-expression of MtACBP2/MsACBP2 also significantly enhanced Arabidopsis tolerance under NaCl and PEG treatments in mature plants. Collectively, our study identified salt and drought responsive ACBP genes in Medicago and verified their functions in increasing resistance against salt and drought stresses.

Serpentine environment prevails over geographic distribution in shaping the genetic diversity of Medicago lupulina L.

Edaphic conditions of serpentine soils, naturally rich in heavy metals, act as a strong selection pressure that shapes specific metal-tolerant ecotypes. Medicago lupulina L. (black medick) is not only a widespread plant species that prefers calcareous and dry soil types but also grows at the borders of serpentine formations. It can also be found in waste and disturbed habitats. This is a species with reported phytoremediation potential, however, there is no published data regarding the impact of the environment on the genetic distribution of this species. The aim of our research was to explore how selection pressure of serpentine soils affects genetic diversity of M. lupulina and to test heavy-metal accumulation capacity of this species. Specimens of 11 M. lupulina populations were collected from serpentine outcrops located in Central and Eastern Bosnia as well as from non-serpentine sites. Soil and plant samples were analyzed for the total contents of heavy metals using air-acetylene flame atomic absorption spectroscopy. Genetic diversity was analyzed using AFLP (Amplified Fragment Length Polymorphism) markers. Serpentine soils showed high nickel, cobalt, chromium and iron concentrations. Nickel and manganese concentrations in soil samples and plant material showed statistically significant correlation. Although plants in two populations show the ability to extract Ni, M. lupulina does not show hyperaccumulating properties. Despite severe selective pressure, genetic diversity in serpentine populations is not reduced. Analyses of intrapopulation and interpopulation genetic diversity showed significant genetic differentiation among populations which is not related to their geographic distance. Population from non-metalliferous soil showed clear separation from all other populations. Diversity data suggest that serpentine populations maintain genetic diversity by undetected mechanisms and that edaphic factors rather than geography influence genetic structure analyzed M. lupulina populations.

Yield of white clover and orchard grass cultivated in mining passives adding black earth and compost as a substrate.

Mining environmental liabilities generate environmental pollution. The objective of the present study was to determine the yield of white clover (Trifolium repens) and orchard grass (Dactylis glomerata) cultivated in mining environmental passives adding black earth and compost as a substrate in the Buenaventura Julcani Huancavelica Company. The treatments were the combinations: 4: 3.1: 3.1: 3:1.1 kg of RP: RP, TN: RP, C: RP, TN, C respectively. They were distributed in four treatments with twelve experimental units for each species of leguminous and gramineous grass, we worked according to the completely randomized design (DCA) with a 2 x 4 factorial arrangement, the experimental unit being a treatment with twelve repetitions. The variables evaluated were: germination percentage (TG) and stem survival percentage (TST). For the statistical analysis, the SPSS software was used.

MrERF039 transcription factor plays an active role in the cold response of Medicago ruthenica as a sugar molecular switch.

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 ß-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.

Spermidine or spermine pretreatment regulates organic metabolites and ions homeostasis in favor of white clover seed germination against salt toxicity.

White clover is widely cultivated as a leguminous forage or ground cover plant worldwide. However, soil salinization decreases its yield and quality. Aims of the present experiment were to elucidate the impact of seed pretreatment with spermidine (Spd) or spermine (Spm) on amylolysis, Na+/K+ accumulation, and metabolic homeostasis during germination. Seed was soaked in distilled water (control), Spd or Spm solution and then germinated under optimal or salt stress conditions for 7 days. Results showed that germination vigor, germination percentage, or seed vigour index of seeds pretreatment with Spd increased by 7%, 11%, or 70% when compared with water-pretreated seeds under salt stress, respectively. Germination percentage or seed vigour index of seeds pretreatment with Spm increased by 17% or 78% than water-pretreated seeds under saline condition, respectively. In response to salt stress, accelerated amylolysis via activation of ß-amylase activity was induced by Spd or Spm pretreatment. Spd or Spm pretreatment also significantly enhanced accumulation of diverse amino acids, organic acids, sugars, and other metabolites (putrescine, myo-inositol, sorbitol, daidzein etc.) associated with enhanced osmotic adjustment, antioxidant capacity, and energy supply during germination under salt stress. In addition, Spd or Spm pretreatment not only significantly reduced salt-induced K+ loss and overaccumulation of Na+, but also improved the ratio of K+ to Na+, contributing to Na+ and K+ balance in seedlings. In response to salt stress, seeds pretreatment with Spd or Spm up-regulated transcription level of NHX2 related to enhancement in compartmentation of Na+ from cytoplasm to vacuole, thus reducing Na+ toxicity in cytoplasm. Spm priming also uniquely up-regulated transcription levels of SKOR, HKT1, and HAL2 associated with K+ and Na + homeostasis and decline in cytotoxicity under salt stress.

Nitrogen offset potential in a multiyear farmlet-scale study: Milk and herbage production from grazed perennial ryegrass-white clover swards.

The objective of this study was to quantify the farm gate nitrogen (N) offset potential of perennial ryegrass (Lolium perenne L.; PRG) white clover (Trifolium repens L.; WC) swards by comparing the herbage and milk production from dairy farmlets that were simulations of full farming systems. A study was established where 120 cows were randomly assigned to 4 farmlets of 10.9 ha (stocking rate: 2.75 cow/ha), composed of 20 paddocks each. Cows were fed 526 kg of DM of concentrate on average each year. The 4 grazing treatments were PRG-only at 150 or 250 kg of N/ha and PRG-WC at 150 or 250 kg of N/ha. Cows remained in their treatment group for an entire grazing season and were re-randomized as they calved across treatments each year. As cows calved in the spring as standard practice in Ireland, they were rotationally grazed from early February both day and night (weather permitting) to mid-November, to a target postgrazing sward height of 4.0 cm. Mean sward WC content was 18.1% and 15.4% for the 150 and 250 kg of N/ha PRG-WC treatments, respectively over the 3-yr period. When WC was included, lowering the N rate did not reduce pregrazing yield, pregrazing height, or herbage removed, but those factors decreased significantly when WC was absent. Total annual herbage DM production was 13,771, 15,242, 14,721, and 15,667 kg of DM/ha for PRG-only swards receiving 150 or 250 kg of N/ha and PRG-WC swards receiving 150 or 250 kg of N/ha, respectively. In addition, when WC was present, compressed postgrazing sward heights were lower (4.10 vs. 4.21 cm) and herbage allowance (approximately 17 kg/cow feed allocation per cow per day) higher than the high-N control (+ 0.7 kg of DM/cow per day). There was a significant increase in milk production, both per cow and per hectare, when WC was included in PRG swards. Over the 3-yr study, cows grazing PRG-WC had greater milk (+304 kg) and milk solids (+31 kg of fat + protein) yields than cows grazing PRG-only swards. This significant increase in milk production suggests that the inclusion of WC in grazing systems can be effectively used to increase milk production per cow and per hectare and help offset nitrogen use. This result shows the potential to increase farm gate N use efficiency and reduce the N surplus compared with PRG-dominant sward grazing systems receiving 250 kg of N/ha, without negatively affecting milk solids yield or herbage production, thus increasing farm profit by €478/ha.

Genomic and Metabolic Characterization of Plant Growth-Promoting Rhizobacteria Isolated from Nodules of Clovers Grown in Non-Farmed Soil.

The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.

Structural Reorganization in Two Alfalfa Mitochondrial Genome Assemblies and Mitochondrial Evolution in Medicago Species.

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution.

Research on the mechanism of plant root protection for soil slope stability.

In order to investigate the impact of herbaceous root development on soil slope stability in expansive soil areas, the research was conducted in the soil slope experimental area of Yaoshi Town, Shangzhou District, Shangluo City. Three types of herbaceous plants, namely Lolium perenne, Medicago, and Cynodon dactylon, were planted to examine their influence on slope stability. The results indicated that Lolium perenne had significantly higher root length density and root surface area density compared to Cynodon dactylon and Medicago. However, the root weight density of Cynodon dactylon was found to be highest. The roots of Lolium perenne, Cynodon dactylon, and Medicago were predominantly observed in diameter ranges of 0 < L ≤ 1.0 mm, 0 < L ≤ 2.5 mm, and 2.5 < L ≤ 3.0 mm, respectively. The roots of herbaceous plants have the ability to enhance water retention in soil, resist hydraulic erosion of slope soil, and reduce soil shrinkage and swelling. During the initial phase of herbaceous planting, there is an accelerated process of organic carbon mineralization in the soil. The roots of herbaceous plants play a crucial role in soil consolidation and slope protection. They achieve this by dispersing large clastic particles, binding small particles together, altering soil porosity, enhancing soil water retention, and reducing soil water infiltration. It was found that Lolium perenne and Medicago, which have well-developed roots, exhibited superior slope protection effects. These findings contribute to the theoretical understanding for the implementation of green ecological protection technology on soil slopes.

Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.