Pollen thermotolerance of a widespread plant, Lotus corniculatus, in response to climate warming: possible local adaptation of populations from different elevations.

One of the most vulnerable phases in the plant life cycle is sexual reproduction, which depends on effective pollen transfer, but also on the thermotolerance of pollen grains. Pollen thermotolerance is temperature-dependent and may be reduced by increasing temperature associated with global warming. A growing body of research has focused on the effect of increased temperature on pollen thermotolerance in crops to understand the possible impact of temperature extremes on yield. Yet, little is known about the effects of temperature on pollen thermotolerance of wild plant species. To fill this gap, we selected Lotus corniculatus s.l. (Fabaceae), a species common to many European habitats and conducted laboratory experiments to test its pollen thermotolerance in response to artificial increase in temperature. To test for possible local adaptation of pollen thermal tolerance, we compared data from six lowland (389-451 m a.s.l.) and six highland (841-1,030 m a.s.l.) populations. We observed pollen germination in vitro at 15 °C, 25 °C, 30 °C, and 40 °C. While lowland plants maintained a stable germination percentage across a broad temperature range (15-30 °C) and exhibited reduced germination only at extremely high temperatures (40 °C), highland plants experienced reduced germination even at 30 °C-temperatures commonly exceeded in lowlands during warm summers. This suggests that lowland populations of L. corniculatus may be locally adapted to higher temperature for pollen germination. On the other hand, pollen tube length decreased with increasing temperature in a similar way in lowland and highland plants. The overall average pollen germination percentage significantly differed between lowland and highland populations, with highland populations displaying higher germination percentage. On the other hand, the average pollen tube length was slightly smaller in highland populations. In conclusion, we found that pollen thermotolerance of L. corniculatus is reduced at high temperature and that the germination of pollen from plant populations growing at higher elevations is more sensitive to increased temperature, which suggests possible local adaptation of pollen thermotolerance.

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.

Interspecific hybridization and inoculation with Pantoea eucalypti improve forage performance of Lotus crop species under alkaline stress.

This study was designed to elucidate the physiological responses of three Lotus forage accessions to alkaline stress, and the influence of inoculating with Pantoea eucalypti endophyte strain on alkaline stress mitigation. A diploid L. corniculatus (Lc) accession, L. tenuis (Lt), and the interspecific hybrid Lt × Lc obtained from these two parental lines were exposed to alkaline stress (pH 8.2). Both Lt and the Lt × Lc hybrid are alkaline-tolerant compared to Lc, based on observations that dry mass was not reduced under stress, and there were no chlorosis symptoms on leaf blades. In all three Lotus accessions, Fe2+ concentration under stress decreased in aerial parts and simultaneously increased in roots. Inoculation with P. eucalypti considerably increased Fe2+ content in shoots of all three Lotus forage species under alkaline treatment. Photochemical efficiency of PSII was affected in Lc accession only when exposed to alkaline treatment. However, when cultivated under alkalinity with inoculation, plants recovered and had photosynthetic parameters equivalent to those in the control treatment. Together, the results highlight the importance of inoculation with P. eucalypti, which contributes significantly to mitigating alkaline stress. All results provide useful information for improving alkaline tolerance traits of Lotus forage species and their interspecific hybrids.

Dynamics of hemoglobins during nodule development, nitrate response, and dark stress in Lotus japonicus.

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.

Balancing nitrate acquisition strategies in symbiotic legumes.

MAIN CONCLUSION: Legumes manage both symbiotic (indirect) and non-symbiotic (direct) nitrogen acquisition pathways. Understanding and optimising the direct pathway for nitrate uptake will support greater legume growth and seed yields. Legumes have multiple pathways to acquire reduced nitrogen to grow and set seed. Apart from the symbiotic N2-fixation pathway involving soil-borne rhizobia bacteria, the acquisition of nitrate and ammonia from the soil can also be an important secondary nitrogen source to meet plant N demand. The balance in N delivery between symbiotic N (indirect) and inorganic N uptake (direct) remains less clear over the growing cycle and with the type of legume under cultivation. In fertile, pH balanced agricultural soils, NO3- is often the predominant form of reduced N available to crop plants and will be a major contributor to whole plant N supply if provided at sufficient levels. The transport processes for NO3- uptake into legume root cells and its transport between root and shoot tissues involves both high and low-affinity transport systems called HATS and LATS, respectively. These proteins are regulated by external NO3- availability and by the N status of the cell. Other proteins also play a role in NO3- transport, including the voltage dependent chloride/nitrate channel family (CLC) and the S-type anion channels of the SLAC/SLAH family. CLC's are linked to NO3- transport across the tonoplast of vacuoles and the SLAC/SLAH's with NO3- efflux across the plasma membrane and out of the cell. An important step in managing the N requirements of a plant are the mechanisms involved in root N uptake and the subsequent cellular distribution within the plant. In this review, we will present the current knowledge of these proteins and what is understood on how they function in key model legumes (Lotus japonicus, Medicago truncatula and Glycine sp.). The review will examine their regulation and role in N signalling, discuss how post-translational modification affects NO3- transport in roots and aerial tissues and its translocation to vegetative tissues and storage/remobilization in reproductive tissues. Lastly, we will present how NO3-influences the autoregulation of nodulation and nitrogen fixation and its role in mitigating salt and other abiotic stresses.

NDR1/HIN1-Like Protein 13 Interacts with Symbiotic Receptor Kinases and Regulates Nodulation in Lotus japonicus.

Lysin-motif receptor-like kinases (LysM-RLKs) are involved in the recognition of microbe-associated molecular patterns to initiate pattern-triggered immunity (PTI). LysM-RLKs are also required for recognition of microbe-derived symbiotic signal molecules upon establishing mutualistic interactions between plants and microsymbionts. A LysM-RLK CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) plays central roles both in chitin-mediated PTI and in arbuscular mycorrhizal symbiosis, suggesting the overlap between immunity and symbiosis, at least in the signal perception and the activation of downstream signal cascades. In this study, we screened for the interacting proteins of Nod factor Receptor1 (NFR1), a CERK1 homolog in the model legume Lotus japonicus, and obtained a protein orthologous to NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13), a protein involved in the activation of innate immunity in Arabidopsis thaliana, which we named LjNHL13a. LjNHL13a interacted with NFR1 and with the symbiosis receptor kinase SymRK. LjNHL13a also displayed positive effects in nodulation. Our results suggest that NHL13 plays a role both in plant immunity and symbiosis, possibly where they overlap. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The CYCLOPS Response Element in the NIN Promoter Is Important but Not Essential for Infection Thread Formation During Lotus japonicus-Rhizobia Symbiosis.

The establishment of the legume-rhizobia symbiosis, termed the root-nodule symbiosis (RNS), requires elaborate interactions at the molecular level. The host plant-derived transcription factor NODULE INCEPTION (NIN) is known to be crucial for RNS, regulating associated processes such as alteration of root hair morphology, infection thread formation, and cell division during nodulation. This emphasizes the importance of the precise spatiotemporal regulation of NIN expression for the establishment of RNS; however, the detailed role of NIN promoter sequences in this process remains unclear. The daphne mutant, a nin mutant allele containing a chromosomal translocation approximately 7 kb upstream of the start codon, does not form nodules but does form infection threads, indicating that the region within 7 kb of the NIN start codon contributes to NIN expression during infection thread formation. CYCLOPS binds to a CYCLOPS response element (CYC-RE) in the NIN promoter, and cyclops mutants are defective in infection thread formation. Here, we performed complementation analysis in nin mutants, using various truncated forms of the NIN promoter, and found that the CYC-RE is important for infection thread formation. Additionally, the CYC-RE deletion mutant, generated through CRISPR/Cas9 technology, displayed a significant reduction in infection thread formation, indicating that the CYC-RE is important for the fine-tuning of NIN expression during this process. However, the fact that infection thread formation is not completely abolished in the CYC-RE deletion mutant suggests that cis and trans factors other than CYCLOPS and the CYC-RE may cooperatively regulate NIN expression for the induction of infection thread formation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The key regulator LcERF056 enhances salt tolerance by modulating reactive oxygen species-related genes in Lotus corniculatus.

BACKGROUND: The APETALA2/ethylene response factor (AP2/ERF) family are important regulatory factors involved in plants' response to environmental stimuli. However, their roles in salt tolerance in Lotus corniculatus remain unclear. RESULTS: Here, the key salt-responsive transcription factor LcERF056 was cloned and characterised. LcERF056 belonging to the B3-1 (IX) subfamily of ERFs was considerably upregulated by salt treatment. LcERF056-fused GFP was exclusively localised to nuclei. Furthermore, LcERF056- overexpression (OE) transgenic Arabidopsis and L. corniculatus lines exhibited significantly high tolerance to salt treatment compared with wild-type (WT) or RNA interference expression (RNAi) transgenic lines at the phenotypic and physiological levels. Transcriptome analysis of OE, RNAi, and WT lines showed that LcERF056 regulated the downstream genes involved in several metabolic pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and yeast one-hybrid (Y1H) assay demonstrated that LcERF056 could bind to cis-element GCC box or DRE of reactive oxygen species (ROS)-related genes such as lipid-transfer protein, peroxidase and ribosomal protein. CONCLUSION: Our results suggested that the key regulator LcERF056 plays important roles in salt tolerance in L. corniculatus by modulating ROS-related genes. Therefore, it may be a useful target for engineering salt-tolerant L. corniculatus or other crops.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota.

Roots of different plant species are colonized by bacterial communities, that are distinct even when hosts share the same habitat. It remains unclear to what extent the host actively selects these communities and whether commensals are adapted to a specific plant species. To address this question, we assembled a sequence-indexed bacterial culture collection from roots and nodules of Lotus japonicus that contains representatives of most species previously identified using metagenomics. We analysed taxonomically paired synthetic communities from L. japonicus and Arabidopsis thaliana in a multi-species gnotobiotic system and detected signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation experiments revealed priority effects during root microbiota assembly, where established communities are resilient to invasion by latecomers, and that host preference of commensal bacteria confers a competitive advantage in their cognate host. Our findings show that host preference in commensal bacteria from diverse taxonomic groups is associated with their invasiveness into standing root-associated communities.

Dysregulation of host-control causes interspecific conflict over host investment into symbiotic organs.

Microbial mutualists provide substantial benefits to hosts that feed back to enhance the fitness of the associated microbes. In many systems, beneficial microbes colonize symbiotic organs, specialized host structures that house symbionts and mediate resources exchanged between parties. Mutualisms are characterized by net benefits exchanged among members of different species, however, inequalities in the magnitude of these exchanges could result in evolutionary conflict, destabilizing the mutualism. We investigated joint fitness effects of root nodule formation, the symbiotic organ of legumes that house nitrogen-fixing rhizobia in planta. We quantified host and symbiont fitness parameters dependent on the number of nodules formed using near-isogenic Lotus japonicus and Mesorhizobium loti mutants, respectively. Empirically estimated fitness functions suggest that legume and rhizobia fitness is aligned as the number of nodules formed increases from zero until the host optimum is reached, a point where aligned fitness interests shift to diverging fitness interests between host and symbiont. However, fitness conflict was only inferred when analyzing wild-type hosts along with their mutants dysregulated for control over nodule formation. These data demonstrate that to avoid conflict, hosts must tightly regulate investment into symbiotic organs maximizing their benefit to cost ratio of associating with microbes.

Responses of Lotus corniculatus to environmental change. 4: Root carbohydrate levels at defoliation and regrowth climatic conditions are major drivers of phenolic content and forage quality.

MAIN CONCLUSION: Differential accumulation of root carbohydrates at defoliation have a higher impact than regrowth environmental conditions on the phenolic content and feed quality of the perennial forage legume Lotus corniculatus. The unpredictable nature of proanthocyanidin (condensed tannin) accumulation in regrowth vegetation of the perennial forage legume Lotus corniculatus represents a dilemma to the wider use of this species in agriculture, and a potential problem in the nutritional ecology of some terrestrial herbivores, as variable condensed tannin levels can result in either beneficial or detrimental effects on animal nutrition. However, the source of this variation has not been extensively explored. High levels of carbon allocation to roots during low-temperature preconditioning of clonal plants were found to significantly increase condensed tannin and flavonol levels in regrowth foliage, while low levels of carbon allocation to roots during periods of high-temperature preconditioning significantly decreased condensed tannin and flavonol levels. Phenolic accumulation and tissue digestibility were also differentially affected by regrowth of these defoliated plants at high CO2 concentrations and by drought. Lower rates of digestion generally paralleled increases in tannin levels in regrowth leaves under the different environmental conditions, with rates of digestion falling in high tannin plants, despite correspondingly higher levels of leaf carbohydrates. Differential accumulation of root carbohydrates between seasons and years may therefore explain some of the variability found in the nutritional quality of the forage of this species.

Endogenous gibberellins affect root nodule symbiosis via transcriptional regulation of NODULE INCEPTION in Lotus japonicus.

Legumes and nitrogen-fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long-distance negative-feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis-acting region on the NIN promoter. Mutants with deletions of this cis-acting region have increased susceptibility to rhizobial infection and reduced GA-induced CLE-RS1 and CLE-RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA-insensitive phenotypes of an AON-defective mutant of HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA-responsive cis-acting region, and subsequently the GA-induced NIN activates the AON system to regulate nodule formation.

Structural signatures in EPR3 define a unique class of plant carbohydrate receptors.

Receptor-mediated perception of surface-exposed carbohydrates like lipo- and exo-polysaccharides (EPS) is important for non-self recognition and responses to microbial associated molecular patterns in mammals and plants. In legumes, EPS are monitored and can either block or promote symbiosis with rhizobia depending on their molecular composition. To establish a deeper understanding of receptors involved in EPS recognition, we determined the structure of the Lotus japonicus (Lotus) exopolysaccharide receptor 3 (EPR3) ectodomain. EPR3 forms a compact structure built of three putative carbohydrate-binding modules (M1, M2 and LysM3). M1 and M2 have unique ßαßß and ßαß folds that have not previously been observed in carbohydrate binding proteins, while LysM3 has a canonical ßααß fold. We demonstrate that this configuration is a structural signature for a ubiquitous class of receptors in the plant kingdom. We show that EPR3 is promiscuous, suggesting that plants can monitor complex microbial communities though this class of receptors.

Submergence tolerance and recovery in Lotus: Variation among fifteen accessions in response to partial and complete submergence.

Several Lotus species are perennial forage legumes which tolerate waterlogging, but knowledge of responses to partial or complete shoot submergence is scant. We evaluated the responses of 15 Lotus accessions to partial and complete shoot submergence and variations in traits associated with tolerance and recovery after de-submergence. Accessions of Lotus tenuis, L. corniculatus, L. pedunculatus and L. japonicus were raised for 43 d and then subjected to aerated root zone (control), deoxygenated stagnant root zone with shoots in air (stagnant), stagnant root zone with partial (75 %) and complete submergence of shoots, for 7 d. The recovery ability from complete submergence was also assessed. We found inter- and intra-specific variations in the stem extension responses (i.e. promoted or restricted compared to controls) depending on water depth. Eight of 15 accessions promoted the stem extension when in partial submergence, while three of those eight (all L. tenuis accessions) had a restricted stem extension when under complete submergence. Two accessions (belonging to L. corniculatus and L. penduculatus species) also promoted the stem extension under complete submergence. The accessions that attained better recovery in terms of leaves produced after de-submergence, were those that had high leaf and root sugar concentration at de-submergence, and high thickness and persistence of gas films on leaves during submergence (all L. tenuis accessions). We conclude that all Lotus accessions were able to tolerate 7 d of partial and complete shoot submergence, despite adopting different stem extension responses.

Novel putative rhizobial species with different symbiovars nodulate Lotus creticus and their differential preference to distinctive soil properties.

Phylogenetically diverse rhizobial strains endemic to Tunisia were isolated from symbiotic nodules of Lotus creticus, growing in different arid extremophile geographical regions of Tunisia, and speciated using multiloci-phylogenetic analysis as Neorhizobium huautlense (LCK33, LCK35, LCO42 and LCO49), Ensifer numidicus (LCD22, LCD25, LCK22 and LCK25), Ensifer meliloti (LCK8, LCK9 and LCK12) and Mesorhizobium camelthorni (LCD11, LCD13, LCD31 and LCD33). In addition, phylogenetic analyses revealed eight additional strains with previously undescribed chromosomal lineages within the genera Ensifer (LCF5, LCF6 and LCF8),Rhizobium (LCF11, LCF12 and LCF14) and Mesorhizobium (LCF16 and LCF19). Analysis using the nodC gene identified five symbiovar groups, four of which were already known. The remaining group composed of two strains (LCD11 and LCD33) represented a new symbiovar of Mesorhizobium camelthorni, which we propose designating as sv. hedysari. Interestingly, we report that soil properties drive and structure the symbiosis of L. creticus and its rhizobia.

Extreme genetic signatures of local adaptation during Lotus japonicus colonization of Japan.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past ~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes.

The Lotus japonicus nucleoporin GLE1 is involved in symbiotic association with rhizobia.

Nucleoporins are components of the nuclear pore complexes, channels that regulate the transport of macromolecules between the nucleus and cytoplasm. The nucleoporin GLE1 (GLFG lethal1) functions in the export of messenger RNAs containing poly(A) tails from the nucleus into the cytoplasm. Here we investigated a mutant of the model legume Lotus japonicus that was defective in GLE1, which we designated Ljgle1. The growth of Ljgle1 was retarded under symbiotic association with rhizobia, and the nitrogen-fixation activities of the nodules were around one-third of those in the wild-type plant. The growth of Ljgle1 was not substantialy recovered by supplemention of combined nitrogen. Nodules formed on the Ljgle1 were smaller than those on the wild-type and colored faint pink. The numbers of infected cells of nodules on the Ljgle1 were smaller than on the wild-type plant, and the former cells remained undeveloped. Rhizobia in the cells of the Ljgle1 exhibited disordered forms, and the symbiosome membrane was closely attached to the bacterial membrane. These results indicate that GLE1 plays a distinct role in the symbiotic association between legumes and rhizobia.

A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus.

Legumes develop root nodules in symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia evoke cell division of differentiated cortical cells into root nodule primordia for accommodating bacterial symbionts. In this study, we show that NODULE INCEPTION (NIN), a transcription factor in Lotus japonicus that is essential for initiating cortical cell divisions during nodulation, regulates the gene ASYMMETRIC LEAVES 2-LIKE 18/LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/LBD16a). Orthologs of ASL18/LBD16a in nonlegume plants are required for lateral root development. Coexpression of ASL18a and the CCAAT box-binding protein Nuclear Factor-Y (NF-Y) subunits, which are also directly targeted by NIN, partially suppressed the nodulation-defective phenotype of L. japonicus daphne mutants, in which cortical expression of NIN was attenuated. Our results demonstrate that ASL18a and NF-Y together regulate nodule organogenesis. Thus, a lateral root developmental pathway is incorporated downstream of NIN to drive nodule symbiosis.

Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.