Floral patterning.

In recent years, flower development has emerged as a model system for studying pattern formation in plants. Homeotic mutants with an altered pattern of floral organs have been found in many species. Recently, several of the floral homeotic genes have been isolated; and the mechanisms underlying pattern formation during flower development are beginning to be elucidated.     

Petal Development in Lotus japonicus

Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral (DV) and organ internal (IN) asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild‐type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA‐like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulating MIXTA‐like gene expression.     

Nodule Organogenesis in Lotus japonicus

Lotus japonicus. Received 25 September 2000/ Accepted in revised form 9 October 2000     

Nitrate assimilation in Lotus japonicus

This paper summarizes some recent advances in the understanding of nitrate assimilation in the model legume Lotus japonicus. First, different types of experimental evidence are presented that emphasize the importance of the root in the nitrate-reducing assimilatory processes in this plant. Secondly, the main results from an ethyl methanesulphonate mutagenesis programme are presented. In this programme, chlorate-resistant and photorespiratory mutants were produced and characterized. The phenotype of one particular chlorate-resistant mutant suggested the importance of a low-affinity nitrate transport system for growth of L. japonicus plants under nitrate nutrition. The phenotype of photorespiratory mutants, affected in all forms of plastid glutamine synthetase in leaves, roots, and nodules, indicated that plastid glutamine synthetase was not required for primary nitrate assimilation nor for the symbiotic associations of the plant (nodulation, mycorrhization), provided photorespiration was suppressed. However, the phenotype of these mutants confirmed that plastid glutamine synthetase was required for the reassimilation of ammonium released by photorespiration. Finally, different aspects of the relationship between nitrate assimilation and osmotic stress in L. japonicus are also discussed, with specific reference to the biosynthesis of proline as an osmolyte.     

Rhizobium-lnduced calcium spiking in Lotus japonicus

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.     

Genome Analysis of Lotus japonicus

Lotus japonicus , a model legume plant, was reviewed and compared with Medicago truncatula and soybean. Several mutant libraries are being analyzed, focusing on the nodulation mechanism. The first plant nodulation gene nin was cloned by Ac-transposon tagging. Soybean remains as the most studied legume, especially in relation to the disease resistance genes. However, Lotus japonicus offers several advantages for molecular genetics, and the remained lackings were recently filled up, namely 1) an appropriate crossing partner for Gifu, accession Miyakojima, was proposed for its 4% polymorphism and smooth recombining ability; 2) a genome library with long inserts, average of 140 kb, and 8.2 genome equivalents of library size, has been established; and 3) the rather low polymorphic rate between Gifu and Miyakojima can be overcome with the HEGS (High Efficiency Genome Scanning). With this infrastructure, positional cloning of the causative genes of several mutant libraries will be accomplished in a short term. Genome sizes of L. japonicus acc. Gifu and Miyakojima were determined with high accuracy, to be 494±0 MB and 512±1 MB, respectively. The feasibility of constructing a physical map of the entire genome, for functional genomics, was discussed. Received 5 September 2000/ Accepted in revised form 11 October 2000     

Phosphoproteome analysis of Lotus japonicus seeds

In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase‐specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM‐kinase target motifs, X‐X‐pS/pT‐Q‐X‐X, were detected in cotyledons. Moreover, by real‐time RT‐PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM‐kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 ( http://proteomecentral.proteomexchange.org/dataset/PXD000053 ).     

Characterization of shade avoidance responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.     

Characterization of Shade Avoidance Responses in Lotus japonicus

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.     

Shoot-applied MeJA suppresses root nodulation in Lotus japonicus

To maintain a symbiotic balance, leguminous plants have a systemic regulatory system called autoregulation of nodulation (AUT). Since AUT is schematically similar to systemic resistance found in plant-pathogen interactions, we examined the effects of methyl jasmonate (MeJA) or methyl salicylate (MeSA) on nodulation in Lotus japonicus. Shoot-applied MeJA strongly suppressed nodulation in the wild type and even hypernodulation in the har1 mutant, whereas MeSA exhibited no effect. MeJA inhibited early stages of nodulation, including infection thread formation and NIN gene expression, and also suppressed lateral root formation. These findings suggest that jasmonic acid and/or its related compounds participate in AUT signaling.     

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus , Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information. Received 29 August 2000/ Accepted in revised form 17 October 2000     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

Lotus japonicus: legume research in the fast lane

Legumes are of immense importance to humanity and a key to sustainable agriculture. Two model species, Lotus japonicus and Medicago truncatula, are the focus of genome sequencing and functional genomics programmes, but most researchers focus exclusively on one or the other. In spite of this, legume researchers now have a unique opportunity to integrate work on these and other legume species, including soybean, common bean and pea to create a platform for comparative genomics second to none of any other plant family. The question is: do we have the scientific fortitude and political will to achieve this?     

LjCYC genes constitute floral dorsoventral asymmetry in Lotus japonicus

Previous study shows that LjCYC2, a CYC-like TCP (TB1, CYC and PCFs) gene in the model legume, Lotus japonicus, is involved in dorsal petal development, which together with the other two homologous genes,LjCYC1 and LjCYC3, belongs to an LjCYC gene cluster. In this report, we modified the transformation system in L. japonicus, and constructed different RNAi transgenes to target different LjCYC genes. The expression of three endogenous LjCYC genes was specifically suppressed by different specific RNAi transgenes, and a chimerical RNAi transgene that contains the specific sequences from LjCYC1 and LjCYC2 was found to downregulate the expression of both endogenous genes simultaneously. Effects of silencing three LjCYC genes were mainly restricted on either dorsal or lateral petals, demonstrating their dorsalizing and lateralizing activities during the development of zygomorphic flower. Furthermore,abolishing the expression of three LjCYC genes could give rise to complete loss of dorsoventral (DV) differentiation in the flower whose petals all resembled the ventral one in the wild type and displayed intact organ internal (IN) asymmetry. Our data demonstrate that during zygomorphic flower development, the DV asymmetry is constituted by the LjCYC genes, while the floral organ IN asymmetry is independently determined by other genetic factors.