DIENELACTONE HYDROLASE LIKE PROTEIN1 negatively regulates the KAI2-ligand pathway in Marchantia polymorpha.

Karrikins are smoke-derived butenolides that induce seed germination and photomorphogenesis in a wide range of plants.1,2,3 KARRIKIN INSENSITIVE2 (KAI2), a paralog of a strigolactone receptor, perceives karrikins or their metabolized products in Arabidopsis thaliana.4,5,6,7 Furthermore, KAI2 is thought to perceive an unidentified plant hormone, called KAI2 ligand (KL).8,9 KL signal is transduced via the interaction between KAI2, MORE AXILLARY GROWTH2 (MAX2), and SUPPRESSOR of MORE AXILLARY GROWTH2 1 LIKE family proteins (SMXLs), followed by the degradation of SMXLs.4,7,10,11,12,13,14 This signaling pathway is conserved both in A. thaliana and the bryophyte Marchantia polymorpha.14 Although the KL signaling pathway is well characterized, the KL metabolism pathways remain poorly understood. Here, we show that DIENELACTONE HYDROLASE LIKE PROTEIN1 (DLP1) is a negative regulator of the KL pathway in M. polymorpha. The KL signal induces DLP1 expression. DLP1 overexpression lines phenocopied the Mpkai2a and Mpmax2 mutants, while dlp1 mutants phenocopied the Mpsmxl mutants. Mutations in the KL signaling genes largely suppressed these phenotypes, indicating that DLP1 acts upstream of the KL signaling pathway, although DLP1 also has KL pathway-independent functions. DLP1 exhibited enzymatic activity toward a potential substrate, suggesting the possibility that DLP1 works through KL inactivation. Investigation of DLP1 homologs in A. thaliana revealed that they do not play a major role in the KL pathway, suggesting different mechanisms for the KL signal regulation. Our findings provide new insights into the regulation of the KL signal in M. polymorpha and the evolution of the KL pathway in land plants.

Activation of Strigolactone Biosynthesis by the DWARF14-LIKE/KARRIKIN-INSENSITIVE2 Pathway in Mycorrhizal Angiosperms, but Not in Arabidopsis, a Non-mycorrhizal Plant.

Strigolactones (SLs) are a class of plant hormones that regulate many aspects of plant growth and development. SLs also improve symbiosis with arbuscular mycorrhizal fungi (AMF) in the rhizosphere. Recent studies have shown that the DWARF14-LIKE (D14L)/KARRIKIN-INSENSITIVE2 (KAI2) family, paralogs of the SL receptor D14, are required for AMF colonization in several flowering plants, including rice. In this study, we found that (-)-GR5, a 2'S-configured enantiomer of a synthetic SL analog (+)-GR5, significantly activated SL biosynthesis in rice roots via D14L. This result is consistent with a recent report, showing that the D14L pathway positively regulates SL biosynthesis in rice. In fact, the SL levels tended to be lower in the roots of the d14l mutant under both inorganic nutrient-deficient and -sufficient conditions. We also show that the increase in SL levels by (-)-GR5 was observed in other mycorrhizal plant species. In contrast, the KAI2 pathway did not upregulate the SL level and the expression of SL biosynthetic genes in Arabidopsis, a non-mycorrhizal plant. We also examined whether the KAI2 pathway enhances SL biosynthesis in the liverwort Marchantia paleacea, where SL functions as a rhizosphere signaling molecule for AMF. However, the SL level and SL biosynthetic genes were not positively regulated by the KAI2 pathway. These results imply that the activation of SL biosynthesis by the D14L/KAI2 pathway has been evolutionarily acquired after the divergence of bryophytes to efficiently promote symbiosis with AMF, although we cannot exclude the possibility that liverworts have specifically lost this regulatory system.

An ancestral function of strigolactones as symbiotic rhizosphere signals.

In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

Functions of Lipids in Development and Reproduction of Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with most land plants. The symbiosis is based on the exchange of nutrients: AMF receive photosynthetically fixed carbon from the plants and deliver mineral nutrients in return. Lipids are important players in the symbiosis. They act as components of the plant-derived membrane surrounding arbuscules, as carbon sources transferred from plants to AMF, as a major form of carbon storage in AMF and as triggers of developmental responses in AMF. In this review, we describe the role of lipids in arbuscular mycorrhizal symbiosis and AMF development.

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

The effects of ERN1 on gene expression during early rhizobial infection in Lotus japonicus.

Legumes develop root nodules in association with compatible rhizobia to overcome nitrogen deficiency. Rhizobia enter the host legume, mainly through infection threads, and induce nodule primordium formation in the root cortex. Multiple transcription factors have been identified to be involved in the regulation of the establishment of root nodule symbiosis, including ERF Required for Nodulation1 (ERN1). ERN1 is involved in a transcription network with CYCLOPS and NODULE INCEPTION (NIN). Mutation of ERN1 often results in misshapen root hair tips, deficient infection thread formation, and immature root nodules. ERN1 directly activates the expression of ENOD11 in Medicago truncatula to assist cell wall remodeling and Epr3 in Lotus japonicus to distinguish rhizobial exopolysaccharide signals. However, aside from these two genes, it remains unclear which genes are regulated by LjERN1 or what role LjERN1 plays during root nodule symbiosis. Thus, we conducted RNA sequencing to compare the gene expression profiles of wild-type L. japonicus and Ljern1-6 mutants. In total, 234 differentially expressed genes were identified as candidate LjERN1 target genes. These genes were found to be associated with cell wall remodeling, signal transduction, phytohormone metabolism, and transcription regulation, suggesting that LjERN1 is involved in multiple processes during the early stages of the establishment of root nodule symbiosis. Many of these candidate genes including RINRK1 showed decreased expression levels in Ljnin-2 mutants based on a search of a public database, suggesting that LjERN1 and LjNIN coordinately regulate gene expression. Our data extend the current understanding of the pleiotropic role of LjERN1 in root nodule symbiosis.

Myristate can be used as a carbon and energy source for the asymbiotic growth of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi, forming symbiotic associations with land plants, are obligate symbionts that cannot complete their natural life cycle without a host. The fatty acid auxotrophy of AM fungi is supported by recent studies showing that lipids synthesized by the host plants are transferred to the fungi, and that the latter lack genes encoding cytosolic fatty acid synthases. Therefore, to establish an asymbiotic cultivation system for AM fungi, we tried to identify the fatty acids that could promote biomass production. To determine whether AM fungi can grow on medium supplied with fatty acids or lipids under asymbiotic conditions, we tested eight saturated or unsaturated fatty acids (C12 to C18) and two ß-monoacylglycerols. Only myristate (C14:0) led to an increase in the biomass of Rhizophagus irregularis, inducing extensive hyphal growth and formation of infection-competent secondary spores. However, such spores were smaller than those generated symbiotically. Furthermore, we demonstrated that R. irregularis can take up fatty acids in its branched hyphae and use myristate as a carbon and energy source. Myristate also promoted the growth of Rhizophagus clarus and Gigaspora margarita Finally, mixtures of myristate and palmitate accelerated fungal growth and induced a substantial change in fatty acid composition of triacylglycerol compared with single myristate application, although palmitate was not used as a carbon source for cell wall biosynthesis in this culture system. Our findings demonstrate that myristate boosts the asymbiotic growth of AM fungi and can also serve as a carbon and energy source.

Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.

Structure-Specific Regulation of Nutrient Transport and Metabolism in Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal fungi (AMF) establish symbiotic relationships with most land plants, mainly for the purpose of nutrient exchange. Many studies have revealed the regulation of processes in AMF, such as nutrient absorption from soil, metabolism and exchange with host plants, and the genes involved. However, the spatial regulation of the genes within the structures comprising each developmental stage is not well understood. Here, we demonstrate the structure-specific transcriptome of the model AMF species, Rhizophagus irregularis. We performed an ultra-low input RNA-seq analysis, SMART-seq2, comparing five extraradical structures, germ tubes, runner hyphae, branched absorbing structures (BAS), immature spores and mature spores. In addition, we reanalyzed the recently reported RNA-seq data comparing intraradical mycelium and arbuscule. Our analyses captured the distinct features of each structure and revealed the structure-specific expression patterns of genes related to nutrient transport and metabolism. Of note, the transcriptional profiles suggest distinct functions of BAS in nutrient absorption. These findings provide a comprehensive dataset to advance our understanding of the transcriptional dynamics of fungal nutrition in this symbiotic system.

Developmental analysis of the early steps in strigolactone-mediated axillary bud dormancy in rice.

By contrast with rapid progress in understanding the mechanisms of biosynthesis and signaling of strigolactone (SL), mechanisms by which SL inhibits axillary bud outgrowth are less well understood. We established a rice (Oryza sativa L.) hydroponic culture system to observe axillary buds at the critical point when the buds enter the dormant state. In situ hybridization analysis indicated that cell division stops in the leaf primordia of the buds entering dormancy. We compared transcriptomes in the axillary buds isolated by laser capture microdissection before and after entering the dormant state and identified genes that are specifically upregulated or downregulated in dormant buds respectively, in SL-mediated axillary bud dormancy. Typically, cell cycle genes and ribosomal genes are included among the active genes while abscisic acid (ABA)-inducible genes are among the dormant genes. Application of ABA to the hydroponic culture suppressed the growth of axillary buds of SL mutants to the same level as wild-type (WT) buds. Tiller number was decreased in the transgenic lines overexpressing OsNCED1, the gene that encodes ABA biosynthesis enzyme. These results indicated that the main site of SL function is the leaf primordia in the axillary bud and that ABA is involved in SL-mediated axillary bud dormancy.

Strigolactone perception and deactivation by a hydrolase receptor DWARF14.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/ß-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis.

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi.

The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi.

BACKGROUND: Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy. RESULTS: In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi. CONCLUSIONS: In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

Strigolactone Biosynthesis Genes of Rice are Required for the Punctual Entry of Arbuscular Mycorrhizal Fungi into the Roots.

Arbuscular mycorrhiza (AM) is a mutualistic association between most plant species and the ancient fungal phylum Glomeromycota in roots, and it plays a key role in a plant's nutrient uptake from the soil. Roots synthesize strigolactones (SLs), derivatives of carotenoids, and exude them to induce energy metabolism and hyphal branching of AM fungi. Despite the well-documented roles of SLs in the pre-symbiotic phase, little is known about the role of SLs in the process of root colonization. Here we show that the expansion of root colonization is suppressed in the mutants of rice (Oryza sativa) SL biosynthesis genes, carotenoid cleavage dioxygenase D10 and more severely in D17. Interestingly, most of the colonization process is normal, i.e. AM fungal hyphae approach the roots and cling around them, and epidermal penetration, arbuscule size, arbuscule number per hyphopodium and metabolic activity of the intraradical mycelium are not affected in d10 and d17 mutants. In contrast, hyphopodium formation is severely attenuated. Our observations establish the requirement for SL biosynthesis genes for efficient hyphopodium formation, suggesting that SLs are required in this process. Efficient hyphopodium formation is required for the punctual internalization of hyphae into roots and maintaining the expansion of colonization.

Spatial regulation of strigolactone function.

Strigolactones are plant hormones that control many aspects of plant development and environmental responses. Despite recent and rapid progress in the biochemical and molecular understanding of strigolactone biosynthesis, transport, and signaling, our knowledge about where strigolactones are produced and where they act is fragmented. In this review, we summarize current knowledge about these aspects of strigolactones, obtained from mutant phenotypes, grafting experiments, gene expression patterns, and protein localization studies. We also discuss the potential of new imaging technologies to reveal the spatial regulation of strigolactone function.

Phloem Transport of the Receptor DWARF14 Protein Is Required for Full Function of Strigolactones.

The cell-to-cell transport of signaling molecules is essential for multicellular organisms to coordinate the action of their cells. Recent studies identified DWARF14 (D14) as a receptor of strigolactones (SLs), molecules that act as plant hormones and inhibit shoot branching. Here, we demonstrate that RAMOSUS3, a pea ortholog of D14, works as a graft-transmissible signal to suppress shoot branching. In addition, we show that D14 protein is contained in phloem sap and transported through the phloem to axillary buds in rice. SLs are not required for the transport of D14 protein. Disruption of D14 transport weakens the suppression of axillary bud outgrowth of rice. Taken together, we conclude that the D14 protein works as an intercellular signaling molecule to fine-tune SL function. Our findings provide evidence that the intercellular transport of a receptor can regulate the action of plant hormones.

Downregulation of rice DWARF 14 LIKE suppress mesocotyl elongation via a strigolactone independent pathway in the dark.

Strigolactones (SLs) are a class of plant hormones that control plant development in response to environmental conditions. In rice, mesocotyl elongation is regulated by SLs in the dark, while mesocotyls are longer in SL deficient or insensitive mutants. SLs are perceived by DWARF14 (D14), which is a member of a small gene family. In this study, we examined the function of another D14 family gene in rice, D14 LIKE (D14L), focusing on mesocotyl growth. The mesocotyls of D14L RNAi lines are longer than those of WT in the dark. This phenotype is enhanced when the D14L RNAi lines are combined with the d14 mutation, suggesting that D14 and D14L work independently to inhibit mesocotyl elongation. This phenotype is alleviated by the exogenous supply of GR24, a synthetic SL, suggesting that D14L is not necessary for SL signaling. D14L mRNA is predominantly expressed in vascular bundles and crown root primordia. Our results suggest that D14L and D14 confer their effects via an SL independent pathway and an SL signaling pathway respectively.

Recent advances in strigolactone research: chemical and biological aspects.

Strigolactones (SLs) are a group of terpenoid lactones that were discovered in the 1960s. They were initially characterized as allelochemicals secreted from roots to the rhizosphere, and have functions in parasitic and symbiotic interactions with root parasitic plants and arbuscular mycorrhizal (AM) fungi, respectively. In 2008, SLs were shown to act as endogenous hormones that regulate shoot branching. The discovery of a hormonal function for SLs has provided a link between genetically studied shoot branching mutants and chemically characterized SLs in earlier studies. This has offered new strategies and experimental tools to address a number of intriguing questions as to the biological function and molecular action of SLs. In this review, we will provide an overview of recent topics on SLs, and highlight new discoveries regarding its biosynthetic pathway and multiple hormonal roles in plant development and adaptive responses.

The D3 F-box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis.

FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice.

Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 microM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 microM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice.