SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus.

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.

Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-binding Proteins in Plants.

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicagotruncatula, involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40. The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicagotruncatula and Lotus japonicus, their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40, as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation.

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

Differentiation between physical and chemical effects of oil presence in freshly spiked soil during rhizoremediation trial.

Petroleum contamination and its remediation via plant-based solutions have got increasing attention by environmental scientists and engineers. In the current study, the physiological and growth responses of two diesel-tolerant plant species (tolerance limit: 1500-2000 mg/kg), Italian ryegrass (Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus), have been investigated in vegetable oil- and diesel oil-amended soils. A long-term (147-day) greenhouse pot experiment was conducted to differentiate the main focus of the study: physical and chemical effects of oil (vegetable and diesel) in freshly spiked soils via evaluating the plant performance and hydrocarbon degradation. Moreover, plant performance was evaluated in terms of seed germination, plant shoot biomass, physiological parameters, and root biomass. Addition of both diesel oil and vegetable oil in freshly spiked soils showed deleterious effects on seedling emergence, root/shoot biomass, and chlorophyll content of grass and legume plants. Italian ryegrass showed more sensitivity in terms of germination rate to both vegetable and diesel oil as compared to non-contaminated soils while Birdsfoot trefoil reduced the germination rate only in diesel oil-impacted soils. The results of the current study suggest that both physical and chemical effects of oil pose negative effects of plant growth and root development. This observation may explain the phenomenon of reduced plant growth in aged/weathered contaminated soils during rhizoremediation experiments.

Transcriptional and Post-transcriptional Modulation of SQU and KEW Activities in the Control of Dorsal-Ventral Asymmetric Flower Development in Lotus japonicus.

In Papilionoideae legume, Lotus japonicus, the development of dorsal-ventral (DV) asymmetric flowers is mainly controlled by two TB1/CYCLOIDEA/PCF (TCP) genes, SQUARED STANDARD (SQU) and KEELED WINGS IN LOTUS (KEW), which determine dorsal and lateral identities, respectively. However, the molecular basis of how these two highly homologous genes orchestrate their diverse functions remains unclear. Here, we analyzed their expression levels, and investigated the transcriptional activities of SQU and KEW. We demonstrated that SQU possesses both activation and repression activities, while KEW acts only as an activator. They form homo- and heterodimers, and then collaboratively regulate their expression at the transcription level. Furthermore, we identified two types of post-transcriptional modifications, phosphorylation and ATP/GTP binding, both of which could affect their transcriptional activities. Mutations in ATP/GTP binding motifs of SQU and KEW lead to failure of phosphorylation, and transgenic plants bearing the mutant proteins display defective DV asymmetric flower development, indicating that the two conjugate modifications are essential for their diverse functions. Altogether, SQU and KEW activities are precisely modulated at both transcription and post-transcription levels, which might link DV asymmetric flower development to different physiological status and/or signaling pathways.

Identification and antioxidant properties of polyphenols in lotus seed epicarp at different ripening stages.

In this study, polyphenols from lotus seed epicarp (PLSE) at three different ripening stages were purified by column chromatography and identified by RP-HPLC and HPLC-ESI-MS(2). The antioxidant activities of PLSE were also investigated. We found that the contents of PLSE at the green ripening stage, half ripening stage and full ripening stage are 13.08%, 10.95% and 6.73% respectively. The levels of catechin, epicatechin, hyperoside, and isoquercitrin in PLSE at the three different ripening stages were different. Moreover, the amounts of catechin and epicatechin decreased, while the contents of hyperoside and isoquercitrin increased as the seed ripened. We found that PLSE at three different ripening stages had good scavenging abilities on DPPH and ABTS(+) radicals. However, the scavenging ability decreased with maturation. Our results may be valuable with regard to the utilization of lotus seed epicarp as a functional food material.

Effects of rapeseed oil on the rhizodegradation of polyaromatic hydrocarbons in contaminated soil.

Plants have the ability to promote degradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil by supporting PAH degrading microorganisms in the rhizosphere (rhizodegradation). The aim of this study was to evaluate if rapeseed oil increases rhizodegradation because various studies have shown that vegetable oils are able to act as extractants for PAHs in contaminated soils and therefore might increase bioavailability of PAHs for microbial degradation. In this study different leguminous and grass species were tested. The results suggested a significant impact of vegetable oil (1 and 3% w/w) on plant growth (decrease of plant height and biomass). The results of the pot experiment showed a decrease in the PAH content of the soil without amendment of rapeseed oil after six months. In soil amended with 1% and 3% of oil, there was no decrease in PAH content within this period. Although no enhancement of PAH degradation by plants could be measured in the bulk soil of the pot experiments, a rhizobox experiment showed a significant reduction of PAH content in the rhizosphere of alfalfa (Medicago sativa cv. Europe). Our investigations also showed significant differences in the degradation behaviour of the 16 individually analysed PAHs.

A MAP kinase kinase interacts with SymRK and regulates nodule organogenesis in Lotus japonicus.

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis.

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus.

• Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. • Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. • The exposure of plants to 10 µM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 µM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. • We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 µM Al.

The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.

The number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.

The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress.

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E(M)) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%. The E(M) values of cortical cells of Lotus roots varied between -115 and -144mV. Treatment with 250microM of AlCl(3) (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51mV (cv. UFGRS) and 16mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K(+) from Al-treated roots (2mM Al) and resulted in a decrease of the diffusion potential (E(D)). The total amount of K(+) in Al-treated roots dropped from 31.4 to 16.8micromolg(-1) FW in sensitive cv. INIA Draco, or from 26.1 to 22.7micromolg(-1) FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21-34% of the control.

Integrative functional genomics of salt acclimatization in the model legume Lotus japonicus.

The model legume Lotus japonicus was subjected to non-lethal long-term salinity and profiled at the ionomic, transcriptomic and metabolomic levels. Two experimental designs with various stress doses were tested: a gradual step acclimatization and an initial acclimatization approach. Ionomic profiling by inductively coupled plasma/atomic emission spectrometry (ICP-AES) revealed salt stress-induced reductions in potassium, phosphorus, sulphur, zinc and molybdenum. Microarray profiling using the Lotus Genechip allowed the identification of 912 probesets that were differentially expressed under the acclimatization regimes. Gas chromatography/mass spectrometry-based metabolite profiling identified 147 differentially accumulated soluble metabolites, indicating a change in metabolic phenotype upon salt acclimatization. Metabolic changes were characterized by a general increase in the steady-state levels of many amino acids, sugars and polyols, with a concurrent decrease in most organic acids. Transcript and metabolite changes exhibited a stress dose-dependent response within the range of NaCl concentrations used, although threshold and plateau behaviours were also observed. The combined observations suggest a successive and increasingly global requirement for the reprogramming of gene expression and metabolic pathways to maintain ionic and osmotic homeostasis. A simple qualitative model is proposed to explain the systems behaviour of plants during salt acclimatization.

Metabolism of reactive oxygen species is attenuated in leghemoglobin-deficient nodules of Lotus japonicus.

Leghemoglobins together with high rates of respiration are believed to be major sources of reactive oxygen species (ROS) in root nodules of leguminous plants. High capacities of antioxidative systems apparently protect this organ from oxidative damage. Using leghemoglobin-RNA interference (LbRNAi) lines of Lotus japonicus, we found that loss of leghemoglobin results in significantly lower H(2)O(2) levels in nodules. Transcript levels and catalytic activities of ascorbate-glutathione cycle enzymes involved in H(2)O(2) detoxification as well as concentrations of reduced ascorbate were also altered in LbRNAi nodules. Thus, symbiotic leghemoglobins contribute significantly to ROS generation in functional nodules.

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides.

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.

Genotype-specific response of a lycaenid herbivore to elevated carbon dioxide and phosphorus availability in calcareous grassland.

Effects of elevated CO2 and P availability on plant growth of the legume Lotus corniculatus and consequences for the butterfly larvae of Polyommatus icarus feeding on L. corniculatus were investigated in screen-aided CO2 control chambers under natural conditions on a calcareous grassland in the Swiss Jura mountains. Elevated CO2 conditions and P fertilisation increased the biomass production of L. corniculatus plants and affected the plant chemical composition. CO2 enrichment increased the C/N ratio and sugar concentration and decreased the N and P concentrations. C- and N-based allelochemicals (cyanoglycosides, total polyphenols and condensed tannins) were only marginally affected by CO2 enrichment. P fertilisation increased the specific leaf area and concentrations of water, N, sugar and P, while the C/N ratio and the concentration of total polyphenols decreased. Furthermore, P availability marginally enhanced the effect of elevated CO2 on the total dry mass and sugar concentration while the opposite occurred for the total polyphenol concentration. The changes in food-plant chemistry as a result of P fertilisation positively affected larval mass gain and accelerated the development time of P. icarus. Only a marginal negative effect on larval mass gain was found for CO2 enrichment. However, we found genotype-specific responses in the development time of P. icarus to elevated CO2 conditions. Larvae originating from different mothers developed better either under elevated CO2 or under ambient CO2 but some did not react to CO2 elevation. As far as we know this is the first finding of a genotype-specific response of an insect herbivore to elevated CO2 which suggests genetic shifts in insect life history traits in response to elevated CO2.

A sucrose transporter, LjSUT4, is up-regulated during Lotus japonicus nodule development.

LjSUT4, encoding a putative sucrose transporter, was identified in a Lotus japonicus nodule cDNA library. The deduced amino acid sequence showed a high degree of identity with sucrose transporters from other plants. Semi-quantitative RT-PCR analysis demonstrated that the L. japonicus SUT4 gene was expressed at high levels in both roots and nodules. In situ hybridization revealed that, in young nodules, SUT4 mRNA transcripts are present in vascular bundles, inner cortex and both infected and uninfected cells while, in mature nodules, accumulation of transcripts was restricted only in vascular bundles and the inner cortex. The results indicated that LjSUT4 codes for a putative sucrose transporter, and its expression pattern suggests a possible shift in the mechanism of sugar transport during nodule development. The role of this polypeptide in sucrose transport and metabolism is discussed.

Isolation of a subfamily of genes for R2R3-MYB transcription factors showing up-regulated expression under nitrogen nutrient-limited conditions.

Plant R2R3-MYB transcription factors are encoded by more than 100 copies of genes. In this study, we attempted to isolate some members of the R2R3-MYB superfamily involved in regulation of nitrogen fixation in legumes. A library of 300 recombinant plasmid clones containing the R2R3-MYB fragments of the superfamily was screened by differential hybridization to isolate R2R3-MYB genes whose expression was up-regulated under nitrogen nutrient-limited conditions. Two groups of clones were identified, each of which seemed to represent a gene responsive to nitrogen starvation. The entire coding regions for the genes were further isolated by PCR and were designated LjMYB101 and LjMYB102. By screening a genomic library of Lotus japonicus with a probe derived from LjMYB101, the third gene, LjMYB103, was isolated. In addition, a candidate for the soybean orthologue of LjMYB101 was isolated and designated GmMYB101. Sequence alignment of the genes with members of the plant R2R3-MYB superfamily showed that they all belonged to the subgroup 10 of the superfamily. The expression analysis of the genes showed that the organ-specific and nitrate-regulated expression profile of MYB101 was very similar to that of CHS in Lotus as well as in soybean, suggesting a possible role for MYB101 in regulation of flavonoid biosynthesis in response to nitrate starvation. On the other hand, an interesting relationship, in structure and function, was found between LjMYB101 and LjGln1, suggesting an alternative role for MYB101 in regulation of nitrogen metabolism.