The Versatile Roles of Type III Secretion Systems in Rhizobium-Legume Symbioses.

To suppress plant immunity and promote the intracellular infection required for fixing nitrogen for the benefit of their legume hosts, many rhizobia use type III secretion systems (T3SSs) that deliver effector proteins (T3Es) inside host cells. As reported for interactions between pathogens and host plants, the immune system of legume hosts and the cocktail of T3Es secreted by rhizobia determine the symbiotic outcome. If they remain undetected, T3Es may reduce plant immunity and thus promote infection of legumes by rhizobia. If one or more of the secreted T3Es are recognized by the cognate plant receptors, defense responses are triggered and rhizobial infection may abort. However, some rhizobial T3Es can also circumvent the need for nodulation (Nod) factors to trigger nodule formation. Here we review the multifaceted roles played by rhizobial T3Es during symbiotic interactions with legumes.

Low-frequency somatic mutations are heritable in tropical trees Dicorynia guianensis and Sextonia rubra.

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and exposure to UV (ultraviolet) radiation increases mutagenesis. To provide insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra, surprisingly almost all found at low frequency. We demonstrate that 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, aging, UV exposure, and mutation rates than commonly thought.

Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

Dancing to a different tune, can we switch from chemical to biological nitrogen fixation for sustainable food security?

Our current food production systems are unsustainable, driven in part through the application of chemically fixed nitrogen. We need alternatives to empower farmers to maximise their productivity sustainably. Therefore, we explore the potential for transferring the root nodule symbiosis from legumes to other crops. Studies over the last decades have shown that preexisting developmental and signal transduction processes were recruited during the evolution of legume nodulation. This allows us to utilise these preexisting processes to engineer nitrogen fixation in target crops. Here, we highlight our understanding of legume nodulation and future research directions that might help to overcome the barrier of achieving self-fertilising crops.

Pan-centromere reveals widespread centromere repositioning of soybean genomes.

Centromere repositioning refers to a de novo centromere formation at another chromosomal position without sequence rearrangement. This phenomenon was frequently encountered in both mammalian and plant species and has been implicated in genome evolution and speciation. To understand the dynamic of centromeres on soybean genome, we performed the pan-centromere analysis using CENH3-ChIP-seq data from 27 soybean accessions, including 3 wild soybeans, 9 landraces, and 15 cultivars. Building upon the previous discovery of three centromere satellites in soybean, we have identified two additional centromere satellites that specifically associate with chromosome 1. These satellites reveal significant rearrangements in the centromere structures of chromosome 1 across different accessions, consequently impacting the localization of CENH3. By comparative analysis, we reported a high frequency of centromere repositioning on 14 out of 20 chromosomes. Most newly emerging centromeres formed in close proximity to the native centromeres and some newly emerging centromeres were apparently shared in distantly related accessions, suggesting their emergence is independent. Furthermore, we crossed two accessions with mismatched centromeres to investigate how centromere positions would be influenced in hybrid genetic backgrounds. We found that a significant proportion of centromeres in the S9 generation undergo changes in size and position compared to their parental counterparts. Centromeres preferred to locate at satellites to maintain a stable state, highlighting a significant role of centromere satellites in centromere organization. Taken together, these results revealed extensive centromere repositioning in soybean genome and highlighted how important centromere satellites are in constraining centromere positions and supporting centromere function.

The productive performance of intercropping.

Crop diversification has been put forward as a way to reduce the environmental impact of agriculture without penalizing its productivity. In this context, intercropping, the planned combination of two or more crop species in one field, is a promising practice. On an average, intercropping saves land compared with the component sole crops, but it remains unclear whether intercropping produces a higher yield than the most productive single crop per unit area, i.e., whether intercropping achieves transgressive overyielding. Here, we quantified the performance of intercropping for the production of grain, calories, and protein in a global meta-analysis of several production indices. The results show that intercrops outperform sole crops when the objective is to achieve a diversity of crop products on a given land area. However, when intercropping is evaluated for its ability to produce raw products without concern for diversity, intercrops on average generate a small loss in grain or calorie yield compared with the most productive sole crop (-4%) but achieve similar or higher protein yield, especially with maize/legume combinations grown at moderate N supply. Overall, although intercropping does not achieve transgressive overyielding on average, our results show that intercropping performs well in producing a diverse set of crop products and performs almost similar to the most productive component sole crop to produce raw products, while improving crop resilience, enhancing ecosystem services, and improving nutrient use efficiency. Our study, therefore, confirms the great interest of intercropping for the development of a more sustainable agricultural production, supporting diversified diets.

A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

Multi-omics analysis reveals the roles of purple acid phosphatases in organic phosphorus utilization by the tropical legume Stylosanthes guianensis.

Stylo (Stylosanthes guianensis) is a tropical legume known for its exceptional tolerance to low phosphate (Pi), a trait believed to be linked to its high acid phosphatase (APase) activity. Previous studies have observed genotypic variations in APase activity in stylo; however, the gene encoding the crucial APase responsible for this variation remains unidentified. In this study, transcriptomic and proteomic analyses were employed to identify eight Pi starvation-inducible (PSI) APases belonging to the purple APase (PAP) family in the roots of stylo and seven in the leaves. Among these PSI-PAPs, SgPAP7 exhibited a significantly positive correlation in its expression levels with the activities of both internal APase and root-associated APase across 20 stylo genotypes under low-Pi conditions. Furthermore, the recombinant SgPAP7 displayed high catalytic activity toward adenosine 5'-diphosphate (ADP) and phosphoenolpyruvate (PEP) in vitro. Overexpression (OE) of SgPAP7 in Arabidopsis facilitated exogenous organic phosphorus utilization. Moreover, SgPAP7 OE lines showed lower shoot ADP and PEP levels than the wild type, implying that SgPAP7 is involved in the catabolism and recycling of endogenous ADP and PEP, which could be beneficial for plant growth in low-Pi soils. In conclusion, SgPAP7 is a key gene with a major role in stylo adaptation to low-Pi conditions by facilitating the utilization of both exogenous and endogenous organic phosphorus sources. It may also function as a PEP phosphatase involved in a glycolytic bypass pathway that minimizes the need for adenylates and Pi. Thus, SgPAP7 could be a promising target for improving tolerance of crops to low-Pi availability.

OROSOMUCOID PROTEIN 1 regulation of sphingolipid synthesis is required for nodulation in Aeschynomene evenia.

Legumes establish symbiotic interactions with nitrogen-fixing rhizobia that are accommodated in root-derived organs known as nodules. Rhizobial recognition triggers a plant symbiotic signaling pathway that activates 2 coordinated processes: infection and nodule organogenesis. How these processes are orchestrated in legume species utilizing intercellular infection and lateral root base nodulation remains elusive. Here, we show that Aeschynomene evenia OROSOMUCOID PROTEIN 1 (AeORM1), a key regulator of sphingolipid biosynthesis, is required for nodule formation. Using A. evenia orm1 mutants, we demonstrate that alterations in AeORM1 function trigger numerous early aborted nodules, defense-like reactions, and shorter lateral roots. Accordingly, AeORM1 is expressed during lateral root initiation and elongation, including at lateral root bases where nodule primordium form in the presence of symbiotic bradyrhizobia. Sphingolipidomics revealed that mutations in AeORM1 lead to sphingolipid overaccumulation in roots relative to the wild type, particularly for very long-chain fatty acid-containing ceramides. Taken together, our findings reveal that AeORM1-regulated sphingolipid homeostasis is essential for rhizobial infection and nodule organogenesis, as well as for lateral root development in A. evenia.

Innovation and appropriation in mycorrhizal and rhizobial Symbioses.

Most land plants benefit from endosymbiotic interactions with mycorrhizal fungi, including legumes and some nonlegumes that also interact with endosymbiotic nitrogen (N)-fixing bacteria to form nodules. In addition to these helpful interactions, plants are continuously exposed to would-be pathogenic microbes: discriminating between friends and foes is a major determinant of plant survival. Recent breakthroughs have revealed how some key signals from pathogens and symbionts are distinguished. Once this checkpoint has been passed and a compatible symbiont is recognized, the plant coordinates the sequential development of two types of specialized structures in the host. The first serves to mediate infection, and the second, which appears later, serves as sophisticated intracellular nutrient exchange interfaces. The overlap in both the signaling pathways and downstream infection components of these symbioses reflects their evolutionary relatedness and the common requirements of these two interactions. However, the different outputs of the symbioses, phosphate uptake versus N fixation, require fundamentally different components and physical environments and necessitated the recruitment of different master regulators, NODULE INCEPTION-LIKE PROTEINS, and PHOSPHATE STARVATION RESPONSES, for nodulation and mycorrhization, respectively.

Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis.

SignificanceOscillations in intracellular calcium concentration play an essential role in the regulation of multiple cellular processes. In plants capable of root endosymbiosis with nitrogen-fixing bacteria and/or arbuscular mycorrhizal fungi, nuclear localized calcium oscillations are essential to transduce the microbial signal. Although the ion channels required to generate the nuclear localized calcium oscillations have been identified, their mechanisms of regulation are unknown. Here, we combined proteomics and engineering approaches to demonstrate that the calcium-bound form of the calmodulin 2 (CaM2) associates with CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15s), closing the channels and providing the negative feedback to sustain the oscillatory mechanism. We further unraveled that the engineered CaM2 accelerates early endosymbioses and enhanced root nodule symbiosis but not arbuscular mycorrhization.

The generality of cryptic dietary niche differences in diverse large-herbivore assemblages.

Ecological niche differences are necessary for stable species coexistence but are often difficult to discern. Models of dietary niche differentiation in large mammalian herbivores invoke the quality, quantity, and spatiotemporal distribution of plant tissues and growth forms but are agnostic toward food plant species identity. Empirical support for these models is variable, suggesting that additional mechanisms of resource partitioning may be important in sustaining large-herbivore diversity in African savannas. We used DNA metabarcoding to conduct a taxonomically explicit analysis of large-herbivore diets across southeastern Africa, analyzing ∼4,000 fecal samples of 30 species from 10 sites in seven countries over 6 y. We detected 893 food plant taxa from 124 families, but just two families-grasses and legumes-accounted for the majority of herbivore diets. Nonetheless, herbivore species almost invariably partitioned food plant taxa; diet composition differed significantly in 97% of pairwise comparisons between sympatric species, and dissimilarity was pronounced even between the strictest grazers (grass eaters), strictest browsers (nongrass eaters), and closest relatives at each site. Niche differentiation was weakest in an ecosystem recovering from catastrophic defaunation, indicating that food plant partitioning is driven by species interactions, and was stronger at low rainfall, as expected if interspecific competition is a predominant driver. Diets differed more between browsers than grazers, which predictably shaped community organization: Grazer-dominated trophic networks had higher nestedness and lower modularity. That dietary differentiation is structured along taxonomic lines complements prior work on how herbivores partition plant parts and patches and suggests that common mechanisms govern herbivore coexistence and community assembly in savannas.

Genetic mapping and transcriptome profiling of a chickpea (Cicer arietinum L.) mutant identifies a novel locus (CaEl) regulating organ size and early vigor.

Chickpea is among the top three legumes produced and consumed worldwide. Early plant vigor, characterized by good germination and rapid seedling growth, is an important agronomic trait in many crops including chickpea, and shows a positive correlation with seed size. In this study, we report a gamma-ray-induced chickpea mutant with a larger organ and seed size. The mutant (elm) exhibits increased early vigor and contains higher proline that contributes to a better tolerance under salt stress at germination, seedling, and early vegetative phase. The trait is governed as monogenic recessive, with wild-type allele being incompletely dominant over the mutant. Genetic mapping of this locus (CaEl) identified it as a previously uncharacterized gene (101503252) in chromosome 1 of the chickpea genome. There is a deletion of this gene in the mutant with a complete loss of expression. In silico analysis suggests that the gene is present as a single copy in chickpea and related legumes of the galegoid clade. In the mutant, cell division and expansion are affected. Transcriptome profiling identified differentially regulated transcripts related to cell division, expansion, cell wall organization, and metabolism in the mutant. The mutant can be exploited in chickpea breeding programs for increasing plant vigor and seed size.

Haplotype-resolved genome of Mimosa bimucronata revealed insights into leaf movement and nitrogen fixation.

BACKGROUND: Mimosa bimucronata originates from tropical America and exhibits distinctive leaf movement characterized by a relative slow speed. Additionally, this species possesses the ability to fix nitrogen. Despite these intriguing traits, comprehensive studies have been hindered by the lack of genomic resources for M. bimucronata. RESULTS: To unravel the intricacies of leaf movement and nitrogen fixation, we successfully assembled a high-quality, haplotype-resolved, reference genome at the chromosome level, spanning 648 Mb and anchored in 13 pseudochromosomes. A total of 32,146 protein-coding genes were annotated. In particular, haplotype A was annotated with 31,035 protein-coding genes, and haplotype B with 31,440 protein-coding genes. Structural variations (SVs) and allele specific expression (ASE) analyses uncovered the potential role of structural variants in leaf movement and nitrogen fixation in M. bimucronata. Two whole-genome duplication (WGD) events were detected, that occurred ~ 2.9 and ~ 73.5 million years ago. Transcriptome and co-expression network analyses revealed the involvement of aquaporins (AQPs) and Ca2+-related ion channel genes in leaf movement. Moreover, we also identified nodulation-related genes and analyzed the structure and evolution of the key gene NIN in the process of symbiotic nitrogen fixation (SNF). CONCLUSION: The detailed comparative genomic and transcriptomic analyses provided insights into the mechanisms governing leaf movement and nitrogen fixation in M. bimucronata. This research yielded genomic resources and provided an important reference for functional genomic studies of M. bimucronata and other legume species.

Comparative phylogenetic analysis of the mediator complex subunit in asparagus bean (Vigna unguiculata ssp. sesquipedialis) and its expression profile under cold stress.

BACKGROUND: The mediator complex subunits (MED) constitutes a multiprotein complex, with each subunit intricately involved in crucial aspects of plant growth, development, and responses to stress. Nevertheless, scant reports pertain to the VunMED gene within the context of asparagus bean (Vigna unguiculata ssp. sesquipedialis). Establishing the identification and exploring the responsiveness of VunMED to cold stress forms a robust foundation for the cultivation of cold-tolerant asparagus bean cultivars. RESULTS: Within this study, a comprehensive genome-wide identification of VunMED genes was executed in the asparagus bean cultivar 'Ningjiang3', resulting in the discovery of 36 distinct VunMED genes. A phylogenetic analysis encompassing 232 MED genes from diverse species, including Arabidopsis, tomatoes, soybeans, mung beans, cowpeas, and asparagus beans, underscored the highly conserved nature of MED gene sequences. Throughout evolutionary processes, each VunMED gene underwent purification and neutral selection, with the exception of VunMED19a. Notably, VunMED9/10b/12/13/17/23 exhibited structural variations discernible across four cowpea species. Divergent patterns of temporal and spatial expression were evident among VunMED genes, with a prominent role attributed to most genes during early fruit development. Additionally, an analysis of promoter cis-acting elements was performed, followed by qRT-PCR assessments on roots, stems, and leaves to gauge relative expression after exposure to cold stress and subsequent recovery. Both treatments induced transcriptional alterations in VunMED genes, with particularly pronounced effects observed in root-based genes following cold stress. Elucidating the interrelationships between subunits involved a preliminary understanding facilitated by correlation and principal component analyses. CONCLUSIONS: This study elucidates the pivotal contribution of VunMED genes to the growth, development, and response to cold stress in asparagus beans. Furthermore, it offers a valuable point of reference regarding the individual roles of MED subunits.

Combining GWAS and comparative genomics to fine map candidate genes for days to flowering in mung bean.

BACKGROUND: Mung bean (Vigna radiata (L.) Wilczek), is an important pulse crop in the global south. Early flowering and maturation are advantageous traits for adaptation to northern and southern latitudes. This study investigates the genetic basis of the Days-to-Flowering trait (DTF) in mung bean, combining genome-wide association studies (GWAS) in mung bean and comparisons with orthologous genes involved with control of DTF responses in soybean (Glycine max (L) Merr) and Arabidopsis (Arabidopsis thaliana). RESULTS: The most significant associations for DTF were on mung bean chromosomes 1, 2, and 4. Only the SNPs on chromosomes 1 and 4 were heavily investigated using downstream analysis. The chromosome 1 DTF association is tightly linked with a cluster of locally duplicated FERONIA (FER) receptor-like protein kinase genes, and the SNP occurs within one of the FERONIA genes. In Arabidopsis, an orthologous FERONIA gene (AT3G51550), has been reported to regulate the expression of the FLOWERING LOCUS C (FLC). For the chromosome 4 DTF locus, the strongest candidates are Vradi04g00002773 and Vradi04g00002778, orthologous to the Arabidopsis PhyA and PIF3 genes, encoding phytochrome A (a photoreceptor protein sensitive to red to far-red light) and phytochrome-interacting factor 3, respectively. The soybean PhyA orthologs include the classical loci E3 and E4 (genes GmPhyA3, Glyma.19G224200, and GmPhyA2, Glyma.20G090000). The mung bean PhyA ortholog has been previously reported as a candidate for DTF in studies conducted in South Korea. CONCLUSION: The top two identified SNPs accounted for a significant proportion (~ 65%) of the phenotypic variability in mung bean DTF by the six significant SNPs (39.61%), with a broad-sense heritability of 0.93. The strong associations of DTF with genes that have orthologs with analogous functions in soybean and Arabidopsis provide strong circumstantial evidence that these genes are causal for this trait. The three reported loci and candidate genes provide useful targets for marker-assisted breeding in mung beans.

Complete chloroplast genome and comparison of herbicides toxicity on Aeschynomene indica (Leguminosae) in upland direct-seeding paddy field.

BACKGROUND: Indian jointvetch (Aeschynomene indica) is a common and pernicious weed found in the upland direct-seeding rice fields in the lower reaches of the Yangtze River in China. However, there are few reports on the degree of harm, genetic characteristics, and management methods of this weed. The purpose of this study is to clarify the harm of Indian jointvetch to upland direct-seeding rice, analyze the genetic characteristics of this weed based on chloroplast genomics and identify its related species, and screen herbicides that are effective in managing this weed in upland direct-seeding rice fields. RESULTS: In a field investigation in upland direct-seeding rice paddies in Shanghai and Jiangsu, we determined that the plant height and maximum lateral distance of Indian jointvetch reached approximately 134.2 cm and 57.9 cm, respectively. With Indian jointvetch present at a density of 4/m2 and 8/m2, the yield of rice decreased by approximately 50% and 70%, respectively. We further obtained the first assembly of the complete chloroplast (cp.) genome sequence of Indian jointvetch (163,613 bp). There were 161 simple sequence repeats, 166 long repeats, and 83 protein-encoding genes. The phylogenetic tree and inverted repeat region expansion and contraction analysis based on cp. genomes demonstrated that species with closer affinity to A. indica included Glycine soja, Glycine max, and Sesbania cannabina. Moreover, a total of 3281, 3840, and 3838 single nucleotide polymorphisms were detected in the coding sequence regions of the cp. genomes of S. cannabina voucher IBSC, G. soja, and G. max compared with the A. indica sequence, respectively. A greenhouse pot experiment indicated that two pre-emergence herbicides, saflufenacil and oxyfluorfen, and two post-emergence herbicides, florpyrauxifen-benzyl and penoxsulam, can more effectively manage Indian jointvetch than other common herbicides in paddy fields. The combination of these two types of herbicides is recommended for managing Indian jointvetch throughout the entire growth period of upland direct-seeding rice. CONCLUSIONS: This study provides molecular resources for future research focusing on the identification of the infrageneric taxa, phylogenetic resolution, and biodiversity of Leguminosae plants, along with recommendations for reliable management methods to control Indian jointvetch.

A Selective Bottleneck During Host Entry Drives the Evolution of New Legume Symbionts.

During the emergence of new host-microbe symbioses, microbial fitness results from the ability to complete the different steps of symbiotic life cycles, where each step imposes specific selective pressures. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts is still poorly known. Here, we characterized the dynamics of phenotypic adaptation to a simplified symbiotic life cycle during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly explained by improved competitiveness for host entry, which outweighed adaptation to within-host proliferation. Whole-population sequencing of bacteria at regular time intervals along this evolution experiment revealed the continuous accumulation of new mutations (fuelled by a transient hypermutagenesis phase occurring at each cycle before host entry, a phenomenon described in previous work) and sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that several adaptive mutations can co-occur in the same cohort. Moreover, all adaptive mutations improved competitiveness for host entry, while only a subset of those also improved within-host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry occurring before within-host proliferation and just after the hypermutagenesis phase in the rhizosphere. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

Evolutionary history of an Irano-Turanian cushion-forming legume (Onobrychis cornuta).

The Irano-Turanian region is one of the largest floristic regions in the world and harbors a high percentage of endemics, including cushion-like and dwarf-shrubby taxa. Onobrychis cornuta is an important cushion-forming element of the subalpine/alpine flora of the Irano-Turanian floristic region. To specify the genetic diversity among the populations of this species (including individuals of O. elymaitica), we employed nrDNA ITS and two noncoding regions of plastid DNA (rpl32-trnL(UAG) and trnT(UGU)-trnL(UAA)). The most striking feature of O. cornuta assemblages was the unexpectedly high nucleotide diversity in both the nDNA and cpDNA dataset. In the analyses of nuclear and plastid regions, 25 ribotypes and 42 haplotypes were found among 77 and 59 accessions, respectively, from Iran, Turkey, and Afghanistan. Network analysis of the datasets demonstrated geographic differentiation within the species. Phylogenetic analyses of all dataset retrieved O. cornuta as a non-monophyletic species due to the inclusion of O. elymaitica, comprising four distinct lineages. In addition, our analyses showed cytonuclear discordance between both nuclear and plastid topologies regarding the position of some O. cornuta individuals. The underlying causes of this inconsistency remain unclear. However, we speculate that chloroplast capture, incomplete lineage sorting, and introgression were the main reasons for this event. Furthermore, molecular dating analysis indicated that O. cornuta originated in the early Pliocene (around 4.8 Mya) and started to diversify throughout the Pliocene and in particular the Pleistocene. Moreover, O. elymaitica was reduced to a subspecific rank within the species.