Intricate intracellular kinase network regulates the Spodoptera lituta-derived elicitor response signaling in Arabidopsis.

Plants defend themselves against herbivores by recognizing herbivore-derived elicitors and activating intracellular signaling. In Arabidopsis, the receptor-like kinase HAK1 recognizes the poly-saccharide elicitor (FrA) from Spodoptera litura larvae, leading to the expression of defense-related genes such as PDF1.2. During this process, the cytoplasmic kinase CRK2 phosphorylates PBL27, triggers the ERF13 expression via ethylene signaling and subsequently leads to PDF1.2 expression. Herein, we investigated four cytoplasmic kinases from the same receptor-like cytoplasmic kinase (RLCK) VII family as PBL27 that interacts with CRK2. Among them, PBL11, like PBL27, is phosphorylated by CRK2 and induces PDF1.2 expression but does not affect ERF13 expression. The weight gain of S. litura larvae on PBL11-deficient mutant plants was only slightly higher than that of wild-type plants, suggesting that PBL11 may function as a minor RLCK that supports the defense response.

Cytoplasmic Kinase Network Mediates Defense Response to Spodoptera litura in Arabidopsis.

Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.

Identification of a new gibberellin receptor agonist, diphegaractin, by a cell-free chemical screening system.

Gibberellin (GA) is a phytohormone that regulates various developmental processes during the plant life cycle. In this study, we identify a new GA agonist, diphegaractin, using a wheat cell-free based drug screening system with grape GA receptor. A GA-dependent interaction assay system using GA receptors and DELLA proteins from Vitis vinifera was constructed using AlphaScreen technology and cell-free produced proteins. From the chemical compound library, diphegaractin was found to enhance the interactions between GA receptors and DELLA proteins from grape in vitro. In grapes, we found that diphegaractin induces elongation of the bunch and increases the sugar concentration of grape berries. Furthermore, diphegaractin shows GA-like activity, including promotion of root elongation in lettuce and Arabidopsis, as well as reducing peel pigmentation and suppressing peel puffing in citrus fruit. To the best of our knowledge, this study is the first to successfully identify a GA receptor agonist showing GA-like activity in agricultural plants using an in vitro molecular-targeted drug screening system.

JUL1, Ring-Type E3 Ubiquitin Ligase, Is Involved in Transcriptional Reprogramming for ERF15-Mediated Gene Regulation.

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

Efficient double-flowered gentian plant production using the CRISPR/Cas9 system.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

De Novo Transcriptome Analysis Reveals Flowering-Related Genes That Potentially Contribute to Flowering-Time Control in the Japanese Cultivated Gentian Gentiana triflora.

Japanese cultivated gentians are perennial plants that flower in early summer to late autumn in Japan, depending on the cultivar. Several flowering-related genes, including GtFT1 and GtTFL1, are known to be involved in regulating flowering time, but many such genes remain unidentified. In this study, we obtained transcriptome profiling data using the Gentiana triflora cultivar 'Maciry', which typically flowers in late July. We conducted deep RNA sequencing analysis using gentian plants grown under natural field conditions for three months before flowering. To investigate diurnal changes, the plants were sampled at 4 h intervals over 24 h. Using these transcriptome data, we determined the expression profiles of leaves based on homology searches against the Flowering-Interactive Database of Arabidopsis. In particular, we focused on transcription factor genes, belonging to the BBX and MADS-box families, and analyzed their developmental and diurnal variation. The expression levels of representative BBX genes were also analyzed under long- and short-day conditions using in-vitro-grown seedlings, and the expression patterns of some BBX genes differed. Clustering analysis revealed that the transcription factor genes were coexpressed with GtFT1. Overall, these expression profiles will facilitate further analysis of the molecular mechanisms underlying the control of flowering time in gentians.

Methods to promote seed germination in the lacquer tree, Toxicodendron vernicifluum (Stokes) F.A. Barkley.

The lacquer tree, Toxicodendron vernicifluum, is a common industrial crop in East Asia. However, T. vernicifluum seeds are extremely difficult to germinate, which poses a major obstacle to establishing seedlings for sap production. In this study, we examined the germination properties of T. vernicifluum seeds in order to establish an inexpensive and effective method to promote seed germination. The seeds are covered with a hard endocarp, which we degrade using conventional sulfuric acid-based methods. Although sulfuric acid was effective in promoting seed germination, the germination rate was less than 5%. In addition to treatment with sulfuric acid, co-treatment with cold temperatures or the phytohormone gibberellic acid increased the germination rate to 22-35%. Seed viability analysis combined with specific gravity-based seed selection revealed that more than half of the seeds housed embryos that were incapable of germination. In additions, specific gravity-based seed selection aided in the selection of seeds capable of germination and improved the germination rate to approximately 47%. Taken together, our results suggest that the low germination rate of T. vernicifluum seeds is due to deep seed dormancy-which is controlled by physical and physiological mechanisms-and low embryo viability. To improve the germination rate of T. vernicifluum seeds, we propose an effective method whereby seeds with good germination capacity are selected based on specific gravity, following which their physiological dormancy is inactivated through cold pretreatment.

Identification of Candidate Genes Responsible for Flower Colour Intensity in Gentiana triflora.

Gentians cultivated in Japan (Gentiana triflora and Gentiana scabra and hybrids) have blue flowers, but flower colour intensity differs among cultivars. The molecular mechanism underlying the variation in flower colour intensity is unclear. Here, we produced F2 progeny derived from an F1 cross of intense- and faint-blue lines and attempted to identify the genes responsible for flower colour intensity using RNA-sequencing analyses. Comparative analysis of flower colour intensity and transcriptome data revealed differentially expressed genes (DEGs), although known flavonoid biosynthesis-related genes showed similar expression patterns. From quantitative RT-PCR (qRT-PCR) analysis, we identified two and four genes with significantly different expression levels in the intense- and faint-blue flower lines, respectively. We conducted further analyses on one of the DEGs, termed GtMIF1, which encodes a putative mini zinc-finger protein homolog, which was most differently expressed in faint-blue individuals. Functional analysis of GtMIF1 was performed by producing stable tobacco transformants. GtMIF1-overexpressing tobacco plants showed reduced flower colour intensity compared with untransformed control plants. DNA-marker analysis also confirmed that the GtMIF1 allele of the faint-blue flower line correlated well with faint flower colour in F2 progeny. These results suggest that GtMIF1 is one of the key genes involved in determining the flower colour intensity of gentian.

Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2;2 to regulate reversible flower opening in Gentiana scabra.

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers.

The rice wound-inducible transcription factor RERJ1 sharing same signal transduction pathway with OsMYC2 is necessary for defense response to herbivory and bacterial blight.

KEY MESSAGE: This study describes biological functions of the bHLH transcription factor RERJ1 involved in the jasmonate response and the related defense-associated metabolic pathways in rice, with particular focus on deciphering the regulatory mechanisms underlying stress-induced volatile emission and herbivory resistance. RERJ1 is rapidly and drastically induced by wounding and jasmonate treatment but its biological function remains unknown as yet. Here we provide evidence of the biological function of RERJ1 in plant defense, specifically in response to herbivory and pathogen attack, and offer insights into the RERJ1-mediated regulation of metabolic pathways of specialized defense compounds, such as monoterpene linalool, in possible collaboration with OsMYC2-a well-known master regulator in jasmonate signaling. In rice (Oryza sativa L.), the basic helix-loop-helix (bHLH) family transcription factor RERJ1 is induced under environmental stresses, such as wounding and drought, which are closely linked to jasmonate (JA) accumulation. Here, we investigated the biological function of RERJ1 in response to biotic stresses, such as herbivory and pathogen infection, using an RERJ1-defective mutant. Transcriptome analysis of the rerj1-Tos17 mutant revealed that RERJ1 regulated the expression of a typical family of conserved JA-responsive genes (e.g., terpene synthases, proteinase inhibitors, and jasmonate ZIM domain proteins). Upon exposure to armyworm attack, the rerj1-Tos17 mutant exhibited more severe damage than the wildtype, and significant weight gain of the larvae fed on the mutant was observed. Upon Xanthomonas oryzae infection, the rerj1-Tos17 mutant developed more severe symptoms than the wildtype. Among RERJ1-regulated terpene synthases, linalool synthase expression was markedly disrupted and linalool emission after wounding was significantly decreased in the rerj1-Tos17 mutant. RERJ1 appears to interact with OsMYC2-a master regulator of JA signaling-and many OsJAZ proteins, although no obvious epistatic interaction was detected between them at the transcriptional level. These results indicate that RERJ1 is involved in the transcriptional induction of JA-mediated stress-responsive genes via physical association with OsMYC2 and mediates defense against herbivory and bacterial infection through JA signaling.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

Expression of LhFT1, the Flowering Inducer of Asiatic Hybrid Lily, in the Bulb Scales.

Asiatic hybrid lily leaves emerge from their bulbs in spring, after cold exposure in winter, and the plant then blooms in early summer. We identified four FLOWERING LOCUS T (FT)-like genes, LhFT1, LhFT4, LhFT6, and LhFT8, from an Asiatic hybrid lily. Floral bud differentiation initiated within bulbs before the emergence of leaves. LhFT genes were mainly expressed in bulb scales, and hardly in leaves, in which the FT-like genes of many plants are expressed in response to environmental signals. LhFT1 was expressed in bulb scales after vernalization and was correlated to flower bud initiation in two cultivars with different flowering behaviors. LhFT8 was upregulated in bulb scales after cold exposure and three alternative splicing variants with a nonsense codon were simultaneously expressed. LhFT6 was upregulated in bulb scales after flower initiation, whereas LhFT4 was expressed constantly in all organs. LhFT1 overexpression complemented the late-flowering phenotype of Arabidopsis ft-10, whereas that of LhFT8 did so partly. LhFT4 and LhFT6 overexpression could not complement. Yeast two-hybrid and in vitro analyses showed that the LhFT1 protein interacted with the LhFD protein. LhFT6 and LhFT8 proteins also interacted with LhFD, as observed in AlphaScreen assay. Based on these results, we revealed that LhFT1 acts as a floral activator during floral bud initiation in Asiatic hybrid lilies. However, the biological functions of LhFT4, LhFT6, and LhFT8 remain unclear.

Soy and Arabidopsis receptor-like kinases respond to polysaccharide signals from Spodoptera species and mediate herbivore resistance.

Plants respond to herbivory by perceiving herbivore danger signal(s) (HDS(s)), including "elicitors", that are present in herbivores' oral secretions (OS) and act to induce defense responses. However, little is known about HDS-specific molecules and intracellular signaling. Here we explored soybean receptor-like kinases (RLKs) as candidates that might mediate HDS-associated RLKs' (HAKs') actions in leaves in response to OS extracted from larvae of a generalist herbivore, Spodoptera litura. Fractionation of OS yielded Frα, which consisted of polysaccharides. The GmHAKs composed of their respective homomultimers scarcely interacted with Frα. Moreover, Arabidopsis HAK1 homomultimers interacted with cytoplasmic signaling molecule PBL27, resulting in herbivory resistance, in an ethylene-dependent manner. Altogether, our findings suggest that HAKs are herbivore-specific RLKs mediating HDS-transmitting, intracellular signaling through interaction with PBL27 and the subsequent ethylene signaling for plant defense responses in host plants.

Tyrosine Kinase-Dependent Defense Responses Against Herbivory in Arabidopsis.

Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants' signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.

The Ring-Type E3 Ubiquitin Ligase JUL1 Targets the VQ-Motif Protein JAV1 to Coordinate Jasmonate Signaling.

Jasmonates regulate plant defense and development. In Arabidopsis (Arabidopsis thaliana), JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1/VQ22) is a repressor of jasmonate-mediated defense responses and is degraded through the ubiquitin-26S proteasome system after herbivory. We found that JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), a RING-type E3 ubiquitin ligase, interacted with JAV1. JUL1 interacted with JAV1 in the nucleus to ubiquitinate JAV1, leading to proteasomal degradation of JAV1. The transcript levels of JUL1 and JAV1 were coordinately and positively regulated by the CORONATINE INSENSITIVE1-dependent signaling pathway in the jasmonate signaling network, but in a manner that was not dependent on CORONATINE INSENSITIVE1-mediated signaling upon herbivory by Spodoptera litura Gain or loss of function of JUL1 modulated the expression levels of the defensin gene PDF1.2 in leaves, conferring on the plants various defense properties against the generalist herbivore S. litura Because neither the JUL1 mutant nor overexpression lines showed any obvious developmental defects, we concluded that the JAV1/JUL1 system functions as a specific coordinator of reprogramming of plant defense responses. Altogether, our findings offer insight into the mechanisms by which the JAV1/JUL1 system acts specifically to coordinate plant defense responses without interfering with plant development or growth.

Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system.

Abscisic acid (ABA) is the main phytohormone involved in abiotic stress response and its adaptation, and is a candidate agrichemical. Consequently, several agonists of ABA have been developed using the yeast two-hybrid system. Here, we describe a novel cell-free-based drug screening approach for the development and validation of ABA receptor agonists. Biochemical validation of this approach between 14 ABA receptors (PYR/PYL/RCARs) and 7 type 2C-A protein phosphatases (PP2CAs) revealed the same interactions as those of previous proteome data, except for nine new interactions. By chemical screening using this approach, we identified two novel ABA receptor agonists, JFA1 (julolidine and fluorine containing ABA receptor activator 1) and JFA2 as its analog. The results of biochemical validation for this approach and biological analysis suggested that JFA1 and JFA2 inhibit seed germination and cotyledon greening of seedlings by activating PYR1 and PYL1, and that JFA2 enhanced drought tolerance without inhibiting root growth by activating not only PYR1 and PYL1 but also PYL5. Thus, our approach was useful for the development of ABA receptor agonists and their validation.

Tyrosine phosphorylation of the GARU E3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradation.

Gibberellin (GA) is a major hormone for plant growth and development. GA response is derived from the degradation of DELLA repressor proteins after GA-dependent complex formation of the GID1 GA receptor with DELLA. Genistein is a known tyrosine (Tyr) kinase inhibitor and inhibits DELLA degradation. However, the biological role of Tyr phosphorylation on the GA response remains unclear. Here, we demonstrate that GARU (GA receptor RING E3 ubiquitin ligase) mediates ubiquitin-dependent degradation of GID1, and that the TAGK2 plant Tyr-kinase is a target of genistein and inhibits GARU-GID1A interactions by phosphorylation of GARU at Tyr321. Genistein induces degradation of GID1 and accumulation of DELLA. Conversely, Arabidopsis garu mutant and TAGK2-overexpressing plants accelerate GID1 stabilization and DELLA degradation. Under salt stress, GARU suppresses seed germination. We propose that GA response is negatively regulated by GARU-dependent GID1 ubiquitination and positively by Tyr phosphorylation of GARU by TAGK2, and genistein inhibits GA signaling by TAGK2 inhibition.Plants respond to gibberellins via GID1-dependent degradation of DELLA proteins. Here, Nemoto et al. show that the gibberellin response is positively regulated by tyrosine phosphorylation of GARU, an E3 ubiquitin ligase that mediates degradation of GID1.

Cell-Free Synthesis of Plant Receptor Kinases.

The wheat germ cell-free protein synthesis system has been used as a eukaryotic protein production system since it was first reported in 1964. Although initially the productivity of this system was not very high, it has now become one of the most versatile protein production systems, thanks to the enhancements made by several groups. In this chapter, we report a protein production method for plant receptor kinases using the wheat cell-free system. We describe a method for the preparation of a cell-free extract from wheat germ, the split-primer PCR method for preparation of transcription templates, and the bilayer cell-free protein synthesis method.

Autophosphorylation Assays Using Plant Receptor Kinases Synthesized in Cell-Free Systems.

The wheat germ cell-free protein synthesis system has a significant advantage for high-throughput production of eukaryotic multidomain proteins in a folded state. In this chapter, we describe two kinds of methods for performing autophosphorylation assay of plant receptor kinases (PRKs) by using the wheat cell-free system. One is an in vitro kinase assay performed using biotin-streptavidin affinity purification technology, and the other is a luminescence-based high-throughput assay for autophosphorylation analysis. We anticipate that our cell-free-based methods might facilitate the characterization of plant PRKs.

OsMYC2, an essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that enhance its transactivation ability.

Biosynthesis of sakuranetin, a flavonoid anti-fungal phytoalexin that occurs in rice, is highly dependent on jasmonic acid (JA) signalling and induced by a variety of environmental stimuli. We previously identified OsNOMT, which encodes naringenin 7-O-methyltransferase (NOMT); NOMT is a key enzyme for sakuranetin production. Although OsNOMT expression is induced by JA treatment, the regulation mechanism that activates the biosynthetic pathway of sakuranetin has not yet been elucidated. In this study, we show that JA-inducible basic helix-loop-helix transcriptional factor OsMYC2 drastically enhances the activity of the OsNOMT promoter and is essential for JA-inducible sakuranetin production. In addition, we identified 2 collaborators of OsMYC2, OsMYC2-like protein 1 and 2 (OsMYL1 and OsMYL2) that further activated the OsNOMT promoter in synergy with OsMYC2. Physical interaction of OsMYC2 with OsMYL1 and OsMYL2 further supported the idea that these interactions lead to the enhancement of the transactivation activity of OsMYC2. Our results indicate that JA signalling via OsMYC2 is reinforced by OsMYL1 and OsMYL2, resulting in the inductive production of sakuranetin during defence responses in rice.