Kohki Akiyama

Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants. The fungi are incapable of completing their life cycle in the absence of a host root. Their spores can germinate and grow in the absence of a host, but their hyphal growth is very limited. Little is known about the molecular mechanisms that govern signalling and recognition between AM fungi and their host plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching in the vicinity of host roots before formation of the appressorium, the structure used to penetrate the plant root. Host roots are known to release signalling molecules that trigger hyphal branching, but these branching factors have not been isolated. Here we have isolated a branching factor from the root exudates of Lotus japonicus and used spectroscopic analysis and chemical synthesis to identify it as a strigolactone, 5-deoxy-strigol. Strigolactones are a group of sesquiterpene lactones, previously isolated as seed-germination stimulants for the parasitic weeds Striga and Orobanche. The natural strigolactones 5-deoxy-strigol, sorgolactone and strigol, and a synthetic analogue, GR24, induced extensive hyphal branching in germinating spores of the AM fungus Gigaspora margarita at very low concentrations.

Inhibition of shoot branching by new terpenoid plant hormones

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class—or their biosynthetic precursors—in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.

Structural Requirements of Strigolactones for Hyphal Branching in AM Fungi

Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C–D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C–D part.

Strigolactones, host recognition signals for root parasitic plants and arbuscular mycorrhizal fungi, from Fabaceae plants

Both root parasitic plants and arbuscular mycorrhizal (AM) fungi take advantage of strigolactones, released from plant roots as signal molecules in the initial communication with host plants, in order to commence parasitism and mutualism, respectively. In this study, strigolactones in root exudates from 12 Fabaceae plants, including hydroponically grown white lupin (Lupinus albus), a nonhost of AM fungi, were characterized by comparing retention times of germination stimulants on reverse-phase high-performance liquid chromatography (HPLC) with those of standards and by using tandem mass spectrometry (LC/MS/MS). All the plant species examined were found to exude known strigolactones, such as orobanchol, orobanchyl acetate, and 5-deoxystrigol, suggesting that these strigolactones are widely distributed in the Fabaceae. It should be noted that even the nonmycotrophic L. albus exuded orobanchol, orobanchyl acetate, 5-deoxystrigol, and novel germination stimulants. By contrast to the mycotrophic Fabaceae plant Trifolium pratense, in which phosphorus deficiency promoted strigolactone exudation, neither phosphorus nor nitrogen deficiency increased exudation of these strigolactones in L. albus. Therefore, the regulation of strigolactone production and/or exudation seems to be closely related to the nutrient acquisition strategy of the plants.

Carlactone is an endogenous biosynthetic precursor for strigolactones

Significance Strigolactones (SLs) were initially characterized as root-derived signals for parasitic and symbiotic interactions with root parasitic plants and arbuscular mycorrhizal fungi, respectively. SLs were later shown to act as endogenous hormones that regulate shoot branching. Carlactone (CL) was identified as a product of three SL biosynthetic enzymes in vitro, and therefore a putative biosynthetic precursor for SLs. However, it was neither detected from plant tissues, nor was the conversion of CL to SL demonstrated in vivo. In this paper, we show that 13C-labeled CL is converted to SLs in vivo, and that endogenous CL is successfully identified from rice and Arabidopsis. These results demonstrate that CL is a true biosynthetic precursor for SLs. Strigolactones (SLs) are a class of terpenoid plant hormones that regulate shoot branching as well as being known as root-derived signals for symbiosis and parasitism. SL has tricyclic-lactone (ABC-ring) and methyl butenolide (D-ring), and they are connected through an enol ether bridge. Recently, a putative biosynthetic intermediate called carlactone (CL), of which carbon skeleton is in part similar to those of SLs, was identified by biochemical analysis of three biosynthetic enzymes, DWARF27, CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), and CCD8 in vitro. However, CL has never been identified from plant tissues, and the conversion of CL to SLs has not been proven in vivo. To address these questions, we chemically synthesized 13C-labeled CL. We show that 13C-labeled CL is converted to (−)-[13C]-2′-epi-5-deoxystrigol ((−)-2′-epi-5DS) and [13C]-orobanchol, endogenous SLs in rice, in the dwarf10 mutant, which is defective in CCD8. In addition, we successfully identified endogenous CL by using liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry in rice and Arabidopsis. Furthermore, we determined the absolute stereochemistry of endogenous CL to be (11R)-configuration, which is the same as that of (−)-2′-epi-5DS at the corresponding position. Feeding experiments showed that only the (11R)-isomer of CL, but not the (11S)-isomer, was converted to (−)-2′-epi-5DS in vivo. Taken together, our data provide conclusive evidence that CL is an endogenous SL precursor that is stereospecifically recognized in the biosynthesis pathway.

Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro.

Significance Strigolactones (SLs) are plant hormones that inhibit shoot branching and are parasitic and symbiotic signals toward root parasitic plants and arbuscular mycorrhizal fungi, respectively. Therefore, the manipulation of SL levels potentially improves the yield of crops. To achieve this goal, the biosynthesis pathway of SLs must be fully understood. SLs are biosynthesized from a precursor, named carlactone (CL), which is derived from carotenoid. However, no downstream pathway of CL has been elucidated. In this study, we show that CL is converted into a carboxylated metabolite, named carlactonoic acid, by Arabidopsis MAX1, the enzymatic function of which had been unknown, and that its methyl ester has the ability to interact with a SL receptor and suppress shoot branching in Arabidopsis. Strigolactones (SLs) stimulate seed germination of root parasitic plants and induce hyphal branching of arbuscular mycorrhizal fungi in the rhizosphere. In addition, they have been classified as a new group of plant hormones essential for shoot branching inhibition. It has been demonstrated thus far that SLs are derived from carotenoid via a biosynthetic precursor carlactone (CL), which is produced by sequential reactions of DWARF27 (D27) enzyme and two carotenoid cleavage dioxygenases CCD7 and CCD8. We previously found an extreme accumulation of CL in the more axillary growth1 (max1) mutant of Arabidopsis, which exhibits increased lateral inflorescences due to SL deficiency, indicating that CL is a probable substrate for MAX1 (CYP711A1), a cytochrome P450 monooxygenase. To elucidate the enzymatic function of MAX1 in SL biosynthesis, we incubated CL with a recombinant MAX1 protein expressed in yeast microsomes. MAX1 catalyzed consecutive oxidations at C-19 of CL to convert the C-19 methyl group into carboxylic acid, 9-desmethyl-9-carboxy-CL [designated as carlactonoic acid (CLA)]. We also identified endogenous CLA and its methyl ester [methyl carlactonoate (MeCLA)] in Arabidopsis plants using LC-MS/MS. Although an exogenous application of either CLA or MeCLA suppressed the growth of lateral inflorescences of the max1 mutant, MeCLA, but not CLA, interacted with Arabidopsis thaliana DWARF14 (AtD14) protein, a putative SL receptor, as shown by differential scanning fluorimetry and hydrolysis activity tests. These results indicate that not only known SLs but also MeCLA are biologically active in inhibiting shoot branching in Arabidopsis.

Confirming Stereochemical Structures of Strigolactones Produced by Rice and Tobacco

Summary Major strigolactones produced by rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) were purified and their stereochemical structures were determined definitely by comparing with optically pure synthetic standards for spectroscopic data.

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.

The Bifunctional Plant Receptor, OsCERK1, Regulates Both Chitin-Triggered Immunity and Arbuscular Mycorrhizal Symbiosis in Rice

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.

New Insecticidal Compounds, Communesins C, D and E, from Penicillium expansum Link MK-57

Three new communesin congeners, communesins C, D, and E, together with two known communesins A and B were isolated from okara that had been fermented with Penicillium expansum Link MK-57. All these communesins showed the insecticidal activity against the silkworms.