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.

Differential amplification of the subtelomeric satellite DNA JcSAT1 in the genus Jatropha L. (Euphorbiaceae).

Satellite DNAs (satDNAs) are highly repetitive sequences that occur in virtually all eukaryotic genomes and can undergo rapid copy number and nucleotide sequence variation among relatives. After chromosomal mapping of the satDNA JcSAT1, it was found a large accumulation at subtelomeres of Jatropha curcas (subgenus Curcas), but an absence of these monomers in J. integerrima (subgenus Jatropha). This fact suggests a dynamic scenario for this satellite repeat in Jatropha genomes. Here, we used a multitasking approach (sequence analysis, DNA blotting and chromosomal mapping) to investigate the molecular organization and chromosomal abundance and distribution of JcSAT1 in a broader group of species from the subgenus Jatropha (J. gossypiifolia, J. mollissima, J. podagrica, and J. multifida) in addition to J. curcas, with the aiming of understanding the evolution of this satDNA. Based on the analysis of BAC clone sequences of J. curcas, a large array (~ 30 kb) of 80 homogeneous monomers of JcSAT1 was identified in BAC 23J11. The monomer size was conserved (~ 358 bp) and contained a telomeric motif at the 5' end. PCR amplification coupled with a Southern blot revealed the presence of JcSAT1-like sequences in all species examined. However, a large set of genome copies was identified only in J. curcas, where a ladder-like pattern with multimers of different sizes was observed. In situ hybridization of BAC 23J11 confirmed the subtelomeric pattern for J. curcas, but showed no signals on chromosomes of species from the subgenus Jatropha. Our data indicate that JcSAT1 is a highly homogeneous satDNA that originated from a region near the telomeres and spread throughout the chromosomal subtermini, possibly due to frequent ectopic recombination between these regions. The abundance of JcSAT1 in the genome of J. curcas suggests that an amplification event occurred either at the base of the subgenus Curcas or at least in this species, although the repeat is shared by all species of the genus studied so far.

Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner.

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix- mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix- phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix- nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1 The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix- nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

Construction of a high-density linkage map and graphical representation of the arrangement of transcriptome-based unigene markers on the chromosomes of onion, Allium cepa L.

BACKGROUND: Genomic information for Allium cepa L. is limited as it is heterozygous and its genome is very large. To elucidate potential SNP markers obtained by NGS, we used a complete set of A. fistulosum L.-A. cepa monosomic addition lines (MALs) and doubled haploids (DHs). These were the parental lines of an A. cepa mapping population for transcriptome-based SNP genotyping. RESULTS: We mapped the transcriptome sequence reads from a series of A. fistulosum-A. cepa MALs onto the unigene sequence of the doubled haploid shallot A. cepa Aggregatum group (DHA) and compared the MAL genotype call for parental bunching onion and shallot transcriptome mapping data. We identified SNP sites with at least four reads on 25,462 unigenes. They were anchored on eight A. cepa chromosomes. A single SNP site was identified on 3,278 unigenes and multiple SNPs were identified on 22,184 unigenes. The chromosome marker information was made public via the web database Allium TDB ( http://alliumtdb.kazusa.or.jp/ ). To apply transcriptome based genotyping approach for genetic mapping, we gathered RNA sequence data from 96 lines of a DHA × doubled haploid bulb onion A. cepa common onion group (DHC) mapping population. After selecting co-dominant SNP sites, 16,872 SNPs were identified in 5,339 unigenes. Of these, at least two SNPs with identical genotypes were found in 1,435 unigenes. We developed a linkage map using genotype information from these unigenes. All unigene markers mapped onto the eight chromosomes and graphical genotyping was conducted based on the unigene order information. Another 2,963 unigenes were allocated onto the eight chromosomes. To confirm the accuracy of this transcriptome-based genetic linkage map, conventional PCR-based markers were used for linkage analysis. All SNP - and PCR-based markers were mapped onto the expected linkage groups and no inconsistency was found among these chromosomal locations. CONCLUSIONS: Effective transcriptome analysis with unique Allium resources successfully associated numerous chromosome markers with unigene information and a high-density A. cepa linkage map. The information on these unigene markers is valuable in genome sequencing and useful trait detection in Allium.

Identification of a Flavin Monooxygenase-Like Flavonoid 8-Hydroxylase with Gossypetin Synthase Activity from Lotus japonicus.

Lotus japonicus is a model legume that accumulates 8-hydroxyflavonol derivatives, such as gossypetin (8-hydroxyquercetin) 3-O-glycoside, which confer the yellow color to its petals. An enzyme, flavonoid 8-hydroxylase (F8H; LjF8H), is assumed to be involved in the biosynthesis, but the specific gene is yet to be identified. The LjF8H cDNA was isolated as a flavin adenine dinucleotide (FAD)-binding monooxygenase-like protein using flower buds and flower-specific EST data of L. japonicus. LjF8H is a single copy gene on chromosome III consisting of six exons. The conserved FAD- and NAD(P)H-dependent oxidase motifs were found in LjF8H. Phylogenetic analysis suggested that LjF8H is a member of the flavin monooxygenase group but distinctly different from other known flavonoid oxygenases. Analysis of recombinant yeast microsome expressing LjF8H revealed that the enzyme catalyzed the 8-hydroxylation of quercetin. Other flavonoids, such as naringenin, eriodictyol, apigenin, luteolin, taxifolin and kaempferol, also acted as substrates of LjF8H. This broad substrate acceptance was unlike known F8Hs in other plants. Interestingly, flavanone and flavanonol, which have saturated C-C bond at positions 2 and 3 of the flavonoid C-ring, produced 6-hyroxylflavonoids as a by-product of the enzymatic reaction. Furthermore, LjF8H only accepted the 2S-isomer of naringenin, suggesting that the conformational state of the substrates might affect product specificity. The overexpression of LjF8H in Arabidopsis thaliana and Petunia hybrida synthesized gossypetin and 8-hydroxykaempferol, respectively, indicating that LjF8H was functional in plant cells. In conclusion, this study represents the first instance of cloning and identification of F8Hs responsible for gossypetin biosynthesis.

Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling.

Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Hemoglobins in the legume-Rhizobium symbiosis.

Legume nodules have two types of hemoglobins: symbiotic or leghemoglobins (Lbs) and nonsymbiotic or phytoglobins (Glbs). The latter are categorized into three phylogenetic classes differing in heme coordination and O2 affinity. This review is focused on the roles of Lbs and Glbs in the symbiosis of rhizobia with crop legumes and the model legumes for indeterminate (Medicago truncatula) and determinate (Lotus japonicus) nodulation. Only two hemoglobin functions are well established in nodules: Lbs deliver O2 to the bacteroids and act as O2 buffers, preventing nitrogenase inactivation; and Glb1-1 modulates nitric oxide concentration during symbiosis, from the early stage, avoiding the plant's defense response, to nodule senescence. Here, we critically examine early and recent results, update and correct the information on Lbs and Glbs with the latest genome versions, provide novel expression data and identify targets for future research. Crucial unresolved questions include the expression of multiple Lbs in nodules, their presence in the nuclei and in uninfected nodule cells, and, intriguingly, their expression in nonsymbiotic tissues. RNA-sequencing data analysis shows that Lbs are expressed as early as a few hours after inoculation and that their mRNAs are also detectable in roots and pods, which clearly suggests that these heme proteins play additional roles unrelated to nitrogen fixation. Likewise, issues awaiting investigation are the functions of other Glbs in nodules, the spatiotemporal expression profiles of Lbs and Glbs at the mRNA and protein levels, and the molecular mechanisms underlying their regulation during nodule development and in response to stress and hormones.

LAZY3 plays a pivotal role in positive root gravitropism in Lotus japonicus.

LAZY1 family genes play important roles in both shoot and root gravitropism in plants. Here we report a Lotus japonicus mutant that displays negative gravitropic response in primary and lateral roots. Map-based cloning identified the mutant gene LAZY3 as a functional ortholog of the LAZY1 gene. Mutation of the LAZY3 gene reduced rootward polar auxin transport (PAT) in the primary root, which was also insensitive to the PAT inhibitor N-1-naphthylphthalamic acid. Moreover, immunolocalization of enhanced green fluorescent protein-tagged LAZY3 in L. japonicus exhibited polar localization of LAZY3 on the plasma membrane in root stele cells. We therefore suggest that the polar localization of LAZY3 in stele cells might be required for PAT in L. japonicus root. LAZY3 transcripts displayed asymmetric distribution at the root tip within hours of gravistimulation, while overexpression of LAZY3 under a constitutive promoter in lazy3 plants rescued the gravitropic response in roots. These data indicate that root gravitropism depends on the presence of LAZY3 but not on its asymmetric expression in root tips. Expression of other LAZY genes in a lazy3 background did not rescue the growth direction of roots, suggesting that the LAZY3 gene plays a distinct role in root gravitropism in L. japonicus.

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.

Loss-of-function of ASPARTIC PEPTIDASE NODULE-INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen-fixing symbiosis with specific Mesorhizobium loti strains.

The nitrogen-fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix- mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix- mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix- mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE-INDUCED 1 (LjAPN1), encodes a nepenthesin-type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain-specific Fix- phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen-fixing) symbiosis in a rhizobial strain-dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.

Exploring Genetic Diversity and Signatures of Horizontal Gene Transfer in Nodule Bacteria Associated with Lotus japonicus in Natural Environments.

To investigate the genetic diversity and understand the process of horizontal gene transfer (HGT) in nodule bacteria associated with Lotus japonicus, we analyzed sequences of three housekeeping and five symbiotic genes using samples from a geographically wide range in Japan. A phylogenetic analysis of the housekeeping genes indicated that L. japonicus in natural environments was associated with diverse lineages of Mesorhizobium spp., whereas the sequences of symbiotic genes were highly similar between strains, resulting in remarkably low nucleotide diversity at both synonymous and nonsynonymous sites. Guanine-cytosine content values were lower in symbiotic genes, and relative frequencies of recombination between symbiotic genes were also lower than those between housekeeping genes. An analysis of molecular variance showed significant genetic differentiation among populations in both symbiotic and housekeeping genes. These results confirm that the Mesorhizobium genes required for symbiosis with L. japonicus behave as a genomic island (i.e., a symbiosis island) and suggest that this island has spread into diverse genomic backgrounds of Mesorhizobium via HGT events in natural environments. Furthermore, our data compilation revealed that the genetic diversity of symbiotic genes in L. japonicus-associated symbionts was among the lowest compared with reports of other species, which may be related to the recent population expansion proposed in Japanese populations of L. japonicus.

LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild-type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp292 to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nm. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons-removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not for Mo delivery to the nodules.

SCARN a Novel Class of SCAR Protein That Is Required for Root-Hair Infection during Legume Nodulation.

Rhizobial infection of legume root hairs requires a rearrangement of the actin cytoskeleton to enable the establishment of plant-made infection structures called infection threads. In the SCAR/WAVE (Suppressor of cAMP receptor defect/WASP family verpolin homologous protein) actin regulatory complex, the conserved N-terminal domains of SCAR proteins interact with other components of the SCAR/WAVE complex. The conserved C-terminal domains of SCAR proteins bind to and activate the actin-related protein 2/3 (ARP2/3) complex, which can bind to actin filaments catalyzing new actin filament formation by nucleating actin branching. We have identified, SCARN (SCAR-Nodulation), a gene required for root hair infection of Lotus japonicus by Mesorhizobium loti. Although the SCARN protein is related to Arabidopsis thaliana SCAR2 and SCAR4, it belongs to a distinct legume-sub clade. We identified other SCARN-like proteins in legumes and phylogeny analyses suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis. Mutation of SCARN reduced formation of infection-threads and their extension into the root cortex and slightly reduced root-hair length. Surprisingly two of the scarn mutants showed constitutive branching of root hairs in uninoculated plants. However we observed no effect of scarn mutations on trichome development or on the early actin cytoskeletal accumulation that is normally seen in root hair tips shortly after M. loti inoculation, distinguishing them from other symbiosis mutations affecting actin nucleation. The C-terminal domain of SCARN binds to ARPC3 and ectopic expression of the N-terminal SCAR-homology domain (but not the full length protein) inhibited nodulation. In addition, we found that SCARN expression is enhanced by M. loti in epidermal cells and that this is directly regulated by the NODULE INCEPTION (NIN) transcription factor.

The LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.

Lotus japonicus cytokinin receptors work partially redundantly to mediate nodule formation.

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.

Nodule Inception creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production.

Autoregulatory negative-feedback loops play important roles in fine-balancing tissue and organ development. Such loops are composed of short-range intercellular signaling pathways via cell-cell communications. On the other hand, leguminous plants use a long-distance negative-feedback system involving root-shoot communication to control the number of root nodules, root lateral organs that harbor symbiotic nitrogen-fixing bacteria known as rhizobia. This feedback system, known as autoregulation of nodulation (AON), consists of two long-distance mobile signals: root-derived and shoot-derived signals. Two Lotus japonicus CLAVATA3/endosperm surrounding region (CLE)-related small peptides, CLE root signal1 (CLE-RS1) and CLE-RS2, function as root-derived signals and are perceived by a shoot-acting AON factor, the hypernodulation aberrant root formation1 (HAR1) receptor protein, an ortholog of Arabidopsis CLAVATA1, which is responsible for shoot apical meristem homeostasis. This peptide-receptor interaction is necessary for systemic suppression of nodulation. How the onset of nodulation activates AON and how optimal nodule numbers are maintained remain unknown, however. Here we show that an RWP-RK-containing transcription factor, nodule inception (NIN), which induces nodule-like structures without rhizobial infection when expressed ectopically, directly targets CLE-RS1 and CLE-RS2. Roots constitutively expressing NIN systemically repress activation of endogenous NIN expression in untransformed roots of the same plant in a HAR1-dependent manner, leading to systemic suppression of nodulation and down-regulation of CLE expression. Our findings provide, to our knowledge, the first molecular evidence of a long-distance autoregulatory negative-feedback loop that homeostatically regulates nodule organ formation.

The Arabidopsis TAC Position Viewer: a high-resolution map of transformation-competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia-0 genome.

We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.