Search

Jasmonic acid (JA) regulates plant growth, development and stress responses. Coronatine insensitive 1 (COI1) and jasmonate zinc-finger inflorescence meristem-domain (JAZ) proteins form a receptor complex for jasmonoyl-l-isoleucine, a biologically active form of JA. Three COIs (OsCOI1a, OsCOI1b and OsCOI2) are encoded in the rice genome. In the present study, we generated mutants for each rice COI gene using genome editing to reveal the physiological functions of the three rice COIs. The oscoi2 mutants, but not the oscoi1a and oscoi1b mutants, exhibited severely low fertility, indicating the crucial role of OsCOI2 in rice fertility. Transcriptomic analysis revealed that the transcriptional changes after methyl jasmonate (MeJA) treatment were moderate in the leaves of oscoi2 mutants compared to those in the wild type or oscoi1a and oscoi1b mutants. MeJA-induced chlorophyll degradation and accumulation of antimicrobial secondary metabolites were suppressed in oscoi2 mutants. These results indicate that OsCOI2 plays a central role in JA response in rice leaves. In contrast, the assessment of growth inhibition upon exogenous application of JA to seedlings of each mutant revealed that rice COIs are redundantly involved in shoot growth, whereas OsCOI2 plays a primary role in root growth. In addition, a co-immunoprecipitation assay showed that OsJAZ2 and OsJAZ5 containing divergent Jas motifs physically interacted only with OsCOI2, whereas OsJAZ4 with a canonical Jas motif interacts with all three rice COIs. The present study demonstrated the functional diversity of rice COIs, thereby providing clues to the mechanisms regulating the various physiological functions of JA.

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g(−1) FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinula edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2′-hydroxypalmitoyl-1-O-β-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], we developed a new method to purify this fungal glucosylceramide using ethanol precipitation and high-performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography–tandem mass spectrometry (LC–MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC–electrospray ionization–MS/MS (LC–ESI–MS/MS).

Laser microdissection (LMD) is used for isolating specific regions or single cells from a wide variety of tissue samples under direct microscopic observation. The LMD method enables the harvest of the cells of interest in a region or specific cells for several analyses, such as DNA/RNA analysis, proteomics, metabolomics, and other molecular analyses. Currently, LMD is used to study various biological events at the tissue or cellular level; it has been used in a wide range of research fields. In this report, we describe techniques for isolating different tissues/specific cells from cryosections of incised Arabidopsis flowering stems by LMD for spatiotemporal quantitative plant hormone analysis. The endogenous indole-3-acetic acid levels in the epidermis/cortex, vascular bundles, and pith of Arabidopsis flowering stems were approximately 19.0 pg mm−3, 33.5 pg mm−3, and 3.32 pg mm−3, respectively, and these endogenous levels were altered spatiotemporally after incision. We also analyzed jasmonic acid from LMD-isolated cells and showed that the endogenous levels increased in the range of approximately 200–3,500 pg mm−3 depending on the tissue and region at 1 h after incision and then decreased to less than 100 pg mm−3 or undetectable levels at 24 h after incision. Quantitative analyses of phytohormones, including jasmonic acid-related molecules, gibberellin, abscisic acid, and cytokinins, could also be performed using the same cell samples. These results showed that spatiotemporal changes in plant hormones could be quantitatively and simultaneously analyzed by LMD-isolated cells from cryosections with positional information. The combination of quantitative analysis by liquid chromatography-mass spectrometry (LC–MS) and sampling by the LMD method provides a comprehensive and quantitative understanding of spatiotemporal changes in plant hormones in a region- and tissue-specific manner. Therefore, LMD-LC–MS methods will contribute to our understanding of the physiological events that control the process of plant growth and development.

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.

KEY MESSAGE We show that in rice, the amino acid-conjugates of JA precursor, OPDA, may function as a non-canonical signal for the production of phytoalexins in coordination with the innate chitin signaling. The core oxylipins, jasmonic acid (JA) and JA-Ile, are well-known as potent regulators of plant defense against necrotrophic pathogens and/or herbivores. However, recent studies also suggest that other oxylipins, including 12-oxo-phytodienoic acid (OPDA), may contribute to plant defense. Here, we used a previously characterized metabolic defense marker, p-coumaroylputrescine (CoP), and fungal elicitor, chitooligosaccharide, to specifically test defense role of various oxylipins in rice (Oryza sativa). While fungal elicitor triggered a rapid production of JA, JA-Ile, and their precursor OPDA, rice cells exogenously treated with the compounds revealed that OPDA, rather than JA-Ile, can stimulate the CoP production. Next, reverse genetic approach and oxylipin-deficient rice mutant (hebiba) were used to uncouple oxylipins from other elicitor-triggered signals. It appeared that, without oxylipins, residual elicitor signaling had only a minimal effect but, in synergy with OPDA, exerted a strong stimulatory activity towards CoP production. Furthermore, as CoP levels were compromised in the OPDA-treated Osjar1 mutant cells impaired in the oxylipin-amino acid conjugation, putative OPDA-amino acid conjugates emerged as hypothetical regulators of CoP biosynthesis. Accordingly, we found several OPDA-amino acid conjugates in rice cells treated with exogenous OPDA, and OPDA-Asp was detected, although in small amounts, in the chitooligosaccharide-treated rice. However, as synthetic OPDA-Asp and OPDA-Ile, so far, failed to induce CoP in cells, it suggests that yet another presumed OPDA-amino acid form(s) could be acting as novel regulator(s) of phytoalexins in rice.

Jasmonic acid and RAP2.6L are induced upon wounding but are not involved in cell proliferation during healing in Arabidopsis hypocotyls. Plants produce jasmonic acid in response to wounding, but its role in healing, if any, has not been determined. Previously, the jasmonic acid-induced transcription factor, RAP2.6L, related to APETALA 2.6-like, was identified as a spatially expressed factor involved in tissue reunion in partially incised flowering stems of Arabidopsis. In the present study, we investigated the function of JA and RAP2.6L on wound healing using an Arabidopsis hypocotyl-grafting system, in which separated tissues are reattached by vascular tissue cell proliferation. The jasmonic acid-responsive genes AOS and JAZ10 were transiently expressed immediately after grafting. We confirmed that the endogenous content of jasmonic acid-Ile, which is the bioactive form of jasmonic acid, increased in hypocotyls 1 h after grafting. Morphological analysis of the grafted tissue revealed that vascular tissue cell proliferation occurred in a similar manner in wild-type Arabidopsis, the jasmonic acid-deficient mutant aos, the jasmonic acid-insensitive mutant coi1, and in Arabidopsis that had been exogenously treated with jasmonic acid. RAP2.6L expression was also induced during graft healing. Because RAP2.6L expression occurred during graft healing in aos and coi1, its expression must be regulated via a jasmonic acid-independent pathway. The rap2.6L mutant and dominant repressor transformants for RAP2.6L showed normal cell proliferation during graft healing. Taken together, our results suggest that JA and RAP2.6L, induced by grafting, are not necessary for cell proliferation process in healing.

Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.

A facile and efficient method has been developed for the optical resolution of racemic jasmonic acid (JA) on a relatively large scale and was successfully utilized for the preparation of optically pure (+)-JA and (−)-JA. We indicated that (+)-JA has lower growth inhibitory activity than (−)-JA in the rice seedling growth test and confirmed in line with an earlier observation that their respective biologically-active forms, (+)-JA-Ile and (−)-JA-Ile, show comparable inhibitory activities. We compared the metabolism of (+)-JA and (−)-JA into (+)-JA-Ile and (−)-JA-Ile, respectively, in the JA-deficient rice cpm2, and found that the exogenously applied (+)-JA was metabolized to the corresponding Ile conjugate less efficiently as compared with (−)-JA. Such metabolic rate difference may cause a discrepancy between biological potencies of (+)-JA and (−)-JA in rice. Abbreviations: FW: fresh weight; Ile: isoleucine; JA: jasmonic acid; JA-Ile: jasmonoyl-l-isoleucine; LC-ESI-MS/MS: liquid chromatography and electrospray ionization tandem mass spectrometry; MeJA: methyl jasmonate; OPDA: 12-oxophytodienoic acid

Rationale Abscisic acid (ABA) and 12-oxo-phytodienoic acid (OPDA) play crucial roles in seed development. However, because of their low ionization efficiencies, visualization by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has been difficult. In this study, we used on-tissue chemical derivatization (OTCD) with the derivatization reagent Girard's T (GirT) in MALDI-IMS to visualize ABA and OPDA. Methods Immature Phaseolus vulgaris L. seeds were homogenized, and frozen homogenate sections were prepared using a cryostat. The concentration of the trifluoroacetic acid (TFA) and spray volume of the GirT solution were optimized using the homogenate sections. Immature seed sections were prepared using a cryostat, and the OTCD efficiency under optimal conditions was measured using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The GirT solution was sprayed on the seed sections, and then MALDI-IMS was performed. Results The optimal TFA concentration and spray volume were 2% and 500 μL, respectively. The OTCD efficiency rates were 61 ± 10% for ABA and 45 ± 5% for OPDA. The peaks corresponding to GirT-derivatized ABA (ABA-GirT) and OPDA (OPDA-GirT) standards were detected on the optimal OTCD-treated seed sections. ABA-GirT was mainly distributed in the embryo, while OPDA-GirT was localized in the external structures. These results are in agreement with our previously published results. Conclusions Our results show that ABA and OPDA in the immature seeds were exactly visualized using OTCD with GirT in MALDI-IMS. Therefore, OTCD with GirT in MALDI-IMS is a promising technique for future research on the biological roles of ABA and OPDA in various immature seeds.

Barnyardgrass (Echinochloa crus-galli) is a pernicious weed in agricultural fields worldwide. The molecular mechanisms underlying its success in the absence of human intervention are presently unknown. Here we report a draft genome sequence of the hexaploid species E. crus-galli, i.e., a 1.27 Gb assembly representing 90.7% of the predicted genome size. An extremely large repertoire of genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with detoxification are found. Two gene clusters involved in the biosynthesis of an allelochemical 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and a phytoalexin momilactone A are found in the E. crus-galli genome, respectively. The allelochemical DIMBOA gene cluster is activated in response to co-cultivation with rice, while the phytoalexin momilactone A gene cluster specifically to infection by pathogenic Pyricularia oryzae. Our results provide a new understanding of the molecular mechanisms underlying the extreme adaptation of the weed., Barnyardgrass is a problematic agricultural weed. Here, via genomic analysis, Guo et al. identify genes potentially underlying its success, including a gene cluster activated in response to co-cultivation with rice that leads to synthesis of the allelochemical DIMBOA.

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl2-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.