ARBUSCULAR MYCORRHIZA-INDUCED KINASES AMK8 and AMK24 associate with the receptor-like kinase KINASE3 to regulate arbuscular mycorrhizal symbiosis in Lotus japonicus.

Arbuscular mycorrhizal (AM) symbiosis is a widespread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3, AMK8, or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171, the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation.

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

NnSnRK1-NnATG1-mediated autophagic cell death governs flower bud abortion in shaded lotus.

Many plants can terminate their flowering process in response to unfavourable environments, but the mechanisms underlying this response are poorly understood. In this study, we observed that the lotus flower buds were susceptible to abortion under shaded conditions. The primary cause of abortion was excessive autophagic cell death (ACD) in flower buds. Blockade of autophagic flux in lotus flower buds consistently resulted in low levels of ACD and improved flowering ability under shaded conditions. Further evidence highlights the importance of the NnSnRK1-NnATG1 signalling axis in inducing ACD in lotus flower buds and culminating in their timely abortion. Under shaded conditions, elevated levels of NnSnRK1 activated NnATG1, which subsequently led to the formation of numerous autophagosome structures in lotus flower bud cells. Excessive autophagy levels led to the bulk degradation of cellular material, which triggered ACD and the abortion of flower buds. NnSnRK1 does not act directly on NnATG1. Other components, including TOR (target of rapamycin), PI3K (phosphatidylinositol 3-kinase) and three previously unidentified genes, appeared to be pivotal for the interaction between NnSnRK1 and NnATG1. This study reveals the role of autophagy in regulating the abortion of lotus flower buds, which could improve reproductive success and act as an energy-efficient measure in plants.

Control of root nodule formation ensures sufficient shoot water availability in Lotus japonicus.

Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability.

Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation.

Auxin methylation by IAMT1, duplicated in the legume lineage, promotes root nodule development in Lotus japonicus.

Legumes attract symbiotic bacteria and create de novo root organs called nodules. Nodule development consists of bacterial infection of root epidermis and subsequent primordium formation in root cortex, steps that need to be spatiotemporally coordinated. The Lotus japonicus mutant "daphne " has uncoupled symbiotic events in epidermis and cortex, in that it promotes excessive bacterial infection in epidermis but does not produce nodule primordia in cortex. Therefore, daphne should be useful for exploring unknown signals that coordinate these events across tissues. Here, we conducted time-course RNA sequencing using daphne after rhizobial infection. We noticed that IAA carboxyl methyltransferase 1 (IAMT1) , which encodes the enzyme that converts auxin (IAA) into its methyl ester (MeIAA), is transiently induced in wild-type roots at early stages of infection but shows different expression dynamics in daphne. IAMT1 serves an important function in shoot development of Arabidopsis, a nonsymbiotic plant, but the function of IAMT1 in roots has not been reported. Phylogenetic tree analysis suggests a gene duplication of IAMT1 in the legume lineage, and we found that one of the two IAMT1s (named IAMT1a) was induced in roots by epidermal infection. IAMT1a knockdown inhibited nodule development in cortex; however, it had no effect on epidermal infection. The amount of root MeIAA increased with rhizobial infection. Application of MeIAA, but not IAA , significantly induced expression of the symbiotic gene NIN in the absence of rhizobial infection. Our results provide evidence for the role of auxin methylation in an early stage of root nodule development.

Tissue-specific regulation of lipid polyester synthesis genes controlling oxygen permeation into Lotus japonicus nodules.

Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a subcompatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically up-regulated in infected compared to noninfected nodules. As hydrophobic modification of cell walls is pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA Reductase genes that were specifically activated in the root and/or in the nodule endodermis. Mutant lines in a Fatty acyl-CoA Reductase gene expressed exclusively in the nodule endodermis had decreased deposition of polyesters on this cell layer and increased nodule permeability compared to wild-type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogenase activity, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules.

Genome-wide identification of the Q-type C2H2 zinc finger protein gene family and expression analysis under abiotic stress in lotus (Nelumbo nucifera G.).

BACKGROUND: Lotus (Nelumbo nucifera G.) is an important aquatic plant with high ornamental, economic, cultural and ecological values, but abiotic stresses seriously affect its growth and distribution. Q-type C2H2 zinc finger proteins (ZFPs) play an important role in plant growth development and environmental stress responses. Although the Q-type C2H2 gene family has been identified in some plants, limited reports has been carried out it in lotus. RESULTS: In this study, we identified 45 Q-type NnZFP members in lotus. Based on the phylogenetic tree, these Q-type NnZFP gene family members were divided into 4 groups, including C1-1i, C1-2i, C1-3i and C1-4i. Promoter cis-acting elements analysis indicated that most Q-type NnZFP gene family members in lotus were associated with response to abiotic stresses. Through collinearity analyses, no tandem duplication gene pairs and 14 segmental duplication gene pairs were identified, which showed that duplication events might play a key role in the expansion of the Q-type NnZFP gene family. The synteny results suggested that 54 and 28 Q-type NnZFP genes were orthologous to Arabidopsis and rice, respectively. The expression patterns of these Q-type NnZFP genes revealed that 30 Q-type NnZFP genes were expressed in at least one lotus tissue. Nn5g30550 showed relatively higher expression levels in all tested tissues. 12 genes were randomly selected with at least one gene from each phylogenetic clade, and the expression of these selected genes were confirmed by qRT-PCR (quantitative real-time polymerase chain reaction). The results indicated that Q-type NnZFP genes were extensively involved in cadmium, drought, salt and cold stresses responses. Among them, 11 genes responded to at least three different stress treatments, especially Nn2g12894, which induced by all four treatments. CONCLUSIONS: These results could increase our understanding of the characterization of the Q-type NnZFP gene family and provide relevant information for further functional analysis of Q-type NnZFP genes in plant development, and abiotic stress tolerance in lotus.

IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss.

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

Comparative plastome analysis and taxonomic classification of snow lotus species (Saussurea, Asteraceae) in Central Asia and Southern Siberia.

Four species of Saussurea, namely S. involucrata, S. orgaadayi, S. bogedaensis, and S. dorogostaiskii, are known as the "snow lotus," which are used as traditional medicines in China (Xinjiang), Kyrgyzstan, Mongolia, and Russia (Southern Siberia). These species are threatened globally, because of illegal harvesting and climate change. Furthermore, the taxonomic classification and identification of these threatened species remain unclear owing to limited research. The misidentification of medicinal species can sometimes be harmful to health. Therefore, the phylogenetic and genomic features of these species need to be confirmed. In this study, we sequenced five complete chloroplast genomes and seven nuclear ITS regions of four snow lotus species and other Saussurea species. We further explored their genetic variety, selective pressure at the sequence level, and phylogenetic relationships using the chloroplast genome, nuclear partial DNA sequences, and morphological features. Plastome of the snow lotus species has a conserved structure and gene content similar to most Saussurea species. Two intergenic regions (ndhJ-ndhK and ndhD-psaC) show significantly high diversity among chloroplast regions. Thus, ITS and these markers are suitable for identifying snow lotus species. In addition, we characterized 43 simple sequence repeats that may be useful in future population genetic studies. Analysis of the selection signatures identified three genes (rpoA, ndhB, and ycf2) that underwent positive selection. These genes may play important roles in the adaptation of the snow lotus species to alpine environments. S. dorogostaiskii is close to S. baicalensis and exhibits slightly different adaptation from others. The taxonomic position of the snow lotus species, confirmed by morphological and molecular evidence, is as follows: (i) S. involucrata has been excluded from the Mongolian flora due to misidentification as S. orgaadayi or S. bogedaensis for a long time; (ii) S. dorogostaiskii belongs to section Pycnocephala subgenus Saussurea, whereas other the snow lotus species belong to section Amphilaena subgenus Amphilaena; and (iii) S. krasnoborovii is synonymous of S. dorogostaiskii. This study clarified the speciation and lineage diversification of the snow lotus species in Central Asia and Southern Siberia.

Unveiling the molecular dynamics of low temperature preservation in postharvest lotus seeds: a transcriptomic perspective.

BACKGROUND: Postharvest quality deterioration poses a significant challenge to the commercial value of fresh lotus seeds. Low temperature storage is widely employed as the primary method for preserving postharvest lotus seeds during storage and transportation. RESULTS: This approach effectively extends the storage life of lotus seeds, resulting in distinct physiological changes compared to room temperature storage, including a notable reduction in starch, protein, H2O2, and MDA content. Here, we conducted RNA-sequencing to generate global transcriptome profiles of postharvest lotus seeds stored under room or low temperature conditions. Principal component analysis (PCA) revealed that gene expression in postharvest lotus seeds demonstrated less variability during low temperature storage in comparison to room temperature storage. A total of 14,547 differentially expressed genes (DEGs) associated with various biological processes such as starch and sucrose metabolism, energy metabolism, and plant hormone signaling response were identified. Notably, the expression levels of DEGs involved in ABA signaling were significantly suppressed in contrast to room temperature storage. Additionally, nine weighted gene co-expression network analysis (WGCNA)-based gene molecular modules were identified, providing insights into the co-expression relationship of genes during postharvest storage. CONCLUSION: Our findings illuminate transcriptional differences in postharvest lotus seeds between room and low temperature storage, offering crucial insights into the molecular mechanisms of low temperature preservation in lotus seeds.

Characterization of DREB family genes in Lotus japonicus and LjDREB2B overexpression increased drought tolerance in transgenic Arabidopsis.

BACKGROUND: Drought stress affects plant growth and development. DREB proteins play important roles in modulating plant growth, development, and stress responses, particularly under drought stress. To study the function of DREB transcription factors (TFs), we screened key DREB-regulating TFs for drought in Lotus japonicus. RESULTS: Forty-two DREB TFs were identified, and phylogenetic analysis of proteins from L. japonicus classified them into five subfamilies (A1, A2, A4, A5, A6). The gene motif composition of the proteins is conserved within the same subfamily. Based on the cis-acting regulatory element analysis, we identified many growth-, hormone-, and stress-responsive elements within the promoter regions of DREB. We further analyzed the expression pattern of four genes in the A2 subfamily in response to drought stress. We found that the expression of most of the LjDREB A2 subfamily genes, especially LjDREB2B, was induced by drought stress. We further generated LjDREB2B overexpression transgenic Arabidopsis plants. Under drought stress, the growth of wild-type (WT) and overexpressing LjDREB2B (OE) Arabidopsis lines was inhibited; however, OE plants showed better growth. The malondialdehyde content of LjDREB2B overexpressing lines was lower than that of the WT plants, whereas the proline content and antioxidant enzyme activities in the OE lines were significantly higher than those in the WT plants. Furthermore, after drought stress, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than those in the WT plants. CONCLUSIONS: Our results facilitate further functional analysis of L. japonicus DREB. LjDREB2B overexpression improves drought tolerance in transgenic Arabidopsis. These results indicate that DREB holds great potential for the genetic improvement of drought tolerance in L. japonicus.

Upregulated synthesis and production of bioactive compounds in Lotus arabicus L. by in vitro feeding with dried powder of date palm seeds.

BACKGROUND: Plants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L. RESULTS: The presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds. CONCLUSION: The marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

NnARF17 and NnARF18 from lotus promote root formation and modulate stress tolerance in transgenic Arabidopsis thaliana.

Auxin response factors (ARFs) play a crucial role in regulating gene expression within the auxin signal transduction pathway, particularly during adventitious root (AR) formation. In this investigation, we identified full-length sequences for ARF17 and ARF18, encompassing 1,800 and 2,055 bp, encoding 599 and 684 amino acid residues, respectively. Despite exhibiting low sequence homology, the ARF17- and ARF18-encoded proteins displayed significant structural similarity and shared identical motifs. Phylogenetic analysis revealed close relationships between NnARF17 and VvARF17, as well as NnARF18 and BvARF18. Both ARF17 and ARF18 demonstrated responsiveness to exogenous indole-3-acetic acid (IAA), ethephon, and sucrose, exhibiting organ-specific expression patterns. Beyond their role in promoting root development, these ARFs enhanced stem growth and conferred drought tolerance while mitigating waterlogging stress in transgenic Arabidopsis plants. RNA sequencing data indicated upregulation of 51 and 75 genes in ARF17 and ARF18 transgenic plants, respectively, including five and three genes associated with hormone metabolism and responses. Further analysis of transgenic plants revealed a significant decrease in IAA content, accompanied by a marked increase in abscisic acid content under normal growth conditions. Additionally, lotus seedlings treated with IAA exhibited elevated levels of polyphenol oxidase, IAA oxidase, and peroxidase. The consistent modulation of IAA content in both lotus and transgenic plants highlights the pivotal role of IAA in AR formation in lotus seedlings.

FER and LecRK show haplotype-dependent cold-responsiveness and mediate freezing tolerance in Lotus japonicus.

Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, FERONIA-receptor like kinase (LjFER) and a S-receptor-like kinase gene (LjLecRK), are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work suggests that recruiting a conserved growth regulator gene, FER, and a receptor-like kinase gene, LecRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering functional genetic insight into perennial herb evolution.

Aromatic amino acid biosynthesis impacts root hair development and symbiotic associations in Lotus japonicus.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

The Medicago SymCEP7 hormone increases nodule number via shoots without compromising lateral root number.

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth.

A pathogenesis-related protein, PRP1, negatively regulates root nodule symbiosis in Lotus japonicus.

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.

Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.