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Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

DOI： 10.1104/pp.19.01420

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Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

DOI： 10.1073/pnas.1721395115

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Plants possess the ability to recognize microbe-associated molecular patterns (MAMPs) and PAMPs through the PRRs, and initiate pattern-triggered immunity. MAMPs are derived from cell-envelope components, secreted materials and cytosolic proteins from bacteria, oomycetes or fungi, and some MAMPs play a similar function in the innate immunity in mammals. Chitin is a representative fungal MAMP and triggers defense signaling in a wide range of plant species. The chitin receptors CEBiP and CERK1 on the plasma membrane have LysM (lysin motif) in their ectodomains. These molecules play an important role for the defense responses in rice and Arabidopsis, strictly recognizing the size and acetylated form of chitin oligosaccharides. However, related LysM receptors also play major roles for the signaling in root nodule and arbuscular mycorrhizal symbiosis. This review summarizes current knowledge on the molecular mechanisms of the defense and symbiosis signaling mediated by LysM receptors, including the activation steps of chitin-induced defense signaling downstream of LysM receptors.

DOI： 10.1177/1753425917738885

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The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca2+ ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca2+ spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

DOI： 10.1111/nph.14539

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In legume-specific rhizobial symbiosis, host plants perceive rhizobial signal molecules, Nod factors, by a pair of LysM receptor-like kinases, NFR1/LYK3 and NFR5/NFP, and activate symbiotic responses through the downstream signaling components also required for arbuscular mycorrhizal (AM) symbiosis. Recently, the rice NFR1/LYK3 ortholog, OsCERK1, was shown to play crucial roles for AM symbiosis. On the other hand, the roles of the NFR5/NFP ortholog in rice have not been elucidated, while it has been shown that NFR5/NFP orthologs, Parasponia PaNFR5 and tomato SlRLK10, engage in AM symbiosis. OsCERK1 also triggers immune responses in combination with a receptor partner, OsCEBiP, against fungal or bacterial infection, thus regulating opposite responses against symbiotic and pathogenic microbes. However, it has not been elucidated how OsCERK1 switches these opposite functions. Here, we analyzed the function of the rice NFR5/NFP ortholog, OsNFR5/OsRLK2, as a possible candidate of the OsCERK1 partner for symbiotic signaling. Inoculation of AM fungi induced the expression of OsNFR5 in the rice root, and the chimeric receptor consisting of the extracellular domain of LjNFR5 and the intracellular domain of OsNFR5 complemented the Ljnfr5 mutant for rhizobial symbiosis, indicating that the intracellular kinase domain of OsNFR5 could activate symbiotic signaling in Lotus japonicus. Although these data suggested the possible involvement of OsNFR5 in AM symbiosis, osnfr5 knockout mutants were colonized by AM fungi similar to the wild-type rice. These observations suggested several possibilities including the presence of functionally redundant genes other than OsNFR5 or involvement of novel ligands, which do not require OsNFR5 for recognition.

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DOI： 10.5511/plantbiotechnology.16.0215a

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The circadian clock plays important roles in the control of photoperiodic flowering in Arabidopsis. Mutations in the LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes (lhy;cca1) accelerate flowering under short days, whereas lhy;cca1 delays flowering under continuous light (LL). The lhy;cca1 mutant also exhibits short hypocotyls and petioles under LL. However, the molecular mechanisms underlying the regulation of both flowering time and organ lengths in the LHY/CCA1-dependent pathway are not fully understood. To address these questions, we performed EMS mutagenesis of the lhy-12;cca1-101 line and screened for mutations that enhance the lhy;cca1 phenotypes under LL. In this screen, we identified a novel allele of dwarf4 (dwf4) and named it petanko 5 (pta5). A similar level of enhancement of the delay in flowering was observed in these two dwf4 mutants when combined with the lhy;cca1 mutations. The lhy;cca1 and dwf4 mutations did not significantly affect the expression level of the floral repressor gene FLC under LL. Our results suggest that a defect in brassinosteroid (BR) signaling delayed flowering independent of the FLC expression level, at least in plants with the lhy;cca1 mutation grown under LL. The dwf4/pta5 mutation did not enhance the late-flowering phenotype of plants overexpressing SVP under LL, suggesting that SVP and BR function in a common pathway that controls flowering time. Our results suggest that the lhy;cca1 mutant exhibits delayed flowering due to both the BR signaling-dependent and -independent pathways under LL.

DOI： 10.5511/plantbiotechnology.16.0601a

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In general, plant growth is inhibited under high-density conditions, while it is promoted under low-density conditions. This is known as the "density effect". Growing plants at high densities is often associated with an accelerated flowering time. Three major pathways [the long day (LD), gibberellic acid (GA), and autonomous/vernalization pathways] are known to play important roles in the control of flowering time. Circadian clock genes, namely, LHY, CCA1, GI, and ELF3, regulate the LD pathway. GAI and FCA control flowering via GA and autonomous pathways, respectively. The density effect on plant size is caused by specific factors such as the amount of nutrition obtained from the soil and touch frequency among plants. However, the molecular mechanism underlying the acceleration of flowering time due to density effects remains unclear. Here, we show the density effects on three Brassicaceae plants, namely, Brassica rapa var. nipposinica, Brassica napus, and Brassica chinensis f. honsaitai. They showed shorter stems and leaves when grown at high densities on soil under continuous light (LL). Shorter stems and leaves, as well as accelerated flowering times, were observed when a model plant, Arabidopsis thaliana, was grown under the same conditions. Unexpectedly, ethylene insensitive 2 (ein2) showed no differences in density effects in our experiments. The acceleration of flowering at higher densities was largely suppressed by gai, but not by gi, lhy;cca1, or fca. These results suggest that the promotion of flowering (as a density effect) is likely dependent on the GA pathway, but not the LD or autonomous pathways.

DOI： 10.5511/plantbiotechnology.16.0622a

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Plant growth promotion and inhibition under low- and high-density conditions (referred to as the density effect) has been studied extensively. Here, we show that such density effects were unaffected by the position of wild-type (WT) and gibberellic acid insensitive (gai) strains of Arabidopsis thaliana (Arabidopsis) within pots. Additionally, petanko 1 (pta1) and pta5 were newly discovered alleles of the ROTUNDIFOLIA 3 (ROT3) and DWARF 4 (DWF4) genes that are involved in brassinosteroid biosynthesis. Unlike gai, the semi-dwarf mutants of pta1 and pta5 exhibited normal flowering times and a shortening of rosette leaves at high densities. Moreover, the pta1 and pta5 variants suppressed flowering stem shortening at high densities. pta5, but not pta1 suppressed the reduction in silique number at intermediate densities. SPINDLY (SPY) is a negative regulator of GA signaling, while PHYTOCHROME B (PHYB) is a red-light photoreceptor. High-density growth did not reduce the flowering time of phyB mutants, but did affect that of spy mutants. Neither spy nor phyB suppressed the shortening of rosette leaves at high densities; however, spy suppressed flowering stem shortening. Moreover, spy suppressed the reduction of silique number at high densities, while and phyB promoted the decrease. These data suggest that GA, BR, and light signaling pathways play important roles in the density effect.

DOI： 10.5511/plantbiotechnology.16.0912a

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Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

DOI： 10.1093/pcp/pcu129

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DOI： 10.5511/plantbiotechnology.13.1104a

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Leguminous plants establish a mutualistic symbiosis with bacteria, collectively referred to as rhizobia. Host plants positively and negatively regulate the symbiotic processes to keep the symbiosis at an appropriate level. Although the plant hormone ethylene is known as a negative regulator of symbiotic processes, the molecular mechanisms of ethylene signaling remain unresolved, especially in the model plant Lotus japonicus. Here, we identified two genes, LjEIN2-1 and LjEIN2-2, from L. japonicus. These genes share moderate similarity in their amino acid sequences, are located on different chromosomes and are composed of different numbers of exons. Suppression of either LjEIN2-1 or LjEIN2-2 expression significantly promoted the root growth of transformed plants on plates containing 1-amino-cyclopropane-carboxylic acid (ACC), the biosynthetic precursor of ethylene. Simultaneous suppression of both LjEIN2-1 and LjEIN2-2 markedly increased the ethylene insensitivity of transgenic roots and resulted in an increased nodulation phenotype. These results indicate that LjEIN2-1 and LjEIN2-2 concertedly regulate ethylene signaling in L. japonicus. We also observed that Nod factor (NF) induced the expression of the ethylene-responsive gene LjACO2, and simultaneous treatment with NF and ACC markedly increases its transcript level compared with either NF or ACC alone. Because LjACO2 encodes ACC oxidase, which is a key enzyme in ethylene biosynthesis, this result suggests the existence of an NF-triggered negative feedback mechanism through ethylene signaling.

DOI： 10.1093/pcp/pct095

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Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO, and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc, and elf3. However, how SVP, FLC, and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated. To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1, and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.

DOI： 10.4161/psb.6.8.16361

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The photoperiodic response is one of the adaptation mechanisms to seasonal changes of lengths of day and night. The circadian clock plays pivotal roles in this process. In Arabidopsis, LHY, CCA1, ELF3, and other clock proteins play major roles in maintaining circadian rhythms. lhy;cca1 double mutants with severe defects in circadian rhythms showed accelerated flowering under short days (SDs), but delayed flowering under continuous light (LL). The protein level of the floral repressor SVP increased in lhy;cca1 mutants under LL, and the late-flowering phenotype of lhy;cca1 mutants was partially suppressed by svp, flc, or elf3. ELF3 interacted with both CCA1 and SVP, and elf3 suppressed the SVP accumulation in lhy;cca1 under LL. These results suggest that the unique mechanism of the inversion of the flowering response of lhy;cca1 under LL may involve both the ELF3-SVP/FLC-dependent and -independent pathways. In this work, elf3-1 seeds were mutagenized with heavy-ion beams and used to identify mutation(s) that delayed flowering under LL but not long days (LDs) or SDs even without ELF3. In this screening, seven candidate lines named suppressor of elf3 1 (self1), sel3, sel5, sel7, sel14, sel15, and sel20 were identified. Genetic analysis indicated that sel20 was a new deletion allele of a mutation in the blue light receptor, CRY2. A late-flowering phenotype and decrease of FT expression in the elf3;sel20 double mutant was obvious under LL but not under SDs or LDs. These results indicated that the late-flowering phenotype in the double mutant elf3;sel20 as well as in lhy;cca1 was affected by the presence of darkness. The results suggest that CRY2 may play more essential roles in the acceleration of flowering under LL than LDs or SDs.

DOI： 10.1093/jxb/erq450

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DOI： 10.1016/B978-0-12-387692-8.00011-4

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DOI： 10.5511/plantbiotechnology.10.0722a

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Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.

DOI： 10.1111/j.1469-8137.2009.02809.x

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DOI： 10.5511/plantbiotechnology.26.217

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DOI： 10.1016/S0065-2296(08)00805-7

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The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.

DOI： 10.1105/tpc.108.061531

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DOI： 10.5511/plantbiotechnology.25.165

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