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Leaf senescence and abscission in autumn are critical phenological events in deciduous woody perennials. After leaf fall, dormant buds remain on deciduous woody perennials, which then enter a winter dormancy phase. Thus, leaf fall is widely believed to be linked to the onset of dormancy. In Rosaceae fruit trees, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factors control bud dormancy. However, apart from their regulatory effects on bud dormancy, the biological functions of DAMs have not been thoroughly characterized. In this study, we revealed a novel DAM function influencing leaf senescence and abscission in autumn. In Prunus mume, PmDAM6 expression was gradually up-regulated in leaves during autumn toward leaf fall. Our comparative transcriptome analysis using two RNA-seq datasets for the leaves of transgenic plants overexpressing PmDAM6 and peach (Prunus persica) DAM6 (PpeDAM6) indicated Prunus DAM6 may up-regulate the expression of genes involved in ethylene biosynthesis and signaling as well as leaf abscission. Significant increases in 1-aminocyclopropane-1-carboxylate accumulation and ethylene emission in DEX-treated 35S:PmDAM6-GR leaves reflect the inductive effect of PmDAM6 on ethylene biosynthesis. Additionally, ethephon treatments promoted autumn leaf senescence and abscission in apple and P. mume, mirroring the changes due to PmDAM6 overexpression. Collectively, these findings suggest that PmDAM6 may induce ethylene emission from leaves, thereby promoting leaf senescence and abscission. This study clarified the effects of Prunus DAM6 on autumn leaf fall, which is associated with bud dormancy onset. Accordingly, in Rosaceae, DAMs may play multiple important roles affecting whole plant growth during the tree dormancy induction phase., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Most deciduous fruit trees cultivated in the temperate zone require a genotype-dependent amounts of chilling exposure for dormancy release and bud break. In Japanese apricot (Prunus mume), DORMANCY-ASSOCIATED MADS-box 6 (PmDAM6) may influence chilling-mediated dormancy release and bud break. In this study, we attempted to elucidate the biological functions of PmDAM6 related to dormancy regulation by analyzing PmDAM6-overexpressing transgenic apple (Malus spp.). We generated 35S:PmDAM6 lines and chemically inducible overexpression lines, 35S:PmDAM6-GR. In both overexpression lines, shoot growth was inhibited and early bud set was observed. In addition, PmDAM6 expression repressed bud break competency during dormancy and delayed bud break. Moreover, PmDAM6 expression increased abscisic acid levels and decreased cytokinins contents during the late dormancy and bud break stages in both 35S:PmDAM6 and 35S:PmDAM6-GR. Our analysis also suggested that abscisic acid levels increased during dormancy but subsequently decreased during dormancy release whereas cytokinins contents increased during the bud break stage in dormant Japanese apricot buds. We previously revealed that PmDAM6 expression is continuously down-regulated during dormancy release toward bud break in Japanese apricot. The PmDAM6 expression pattern was concurrent with a decrease and increase in the abscisic acid and cytokinins contents, respectively, in dormant Japanese apricot buds. Therefore, we hypothesize that PmDAM6 represses the bud break competency during dormancy and bud break stages in Japanese apricot by modulating abscisic acid and cytokinins accumulation in dormant buds., Competing Interests: The authors have declared that no competing interests exist.

Phytocassanes and momilactones are known as major diterpenoid phytoalexins (DPs), characterized by abundant production and antimicrobial activity, and their biosynthetic genes are clustered in rice genomes. The basic leucine zipper transcription factor OsTGAP1 is known to act as a regulator of the coordinated production of DPs in cultured rice cells, but in planta functions of OsTGAP1 remain largely unknown. Here, we present evidence on the biological function of OsTGAP1 in planta. In wild-type plants, OsTGAP1 is abundantly expressed in roots compared with that in shoots. Moreover, the inductive expression of OsTGAP1 under jasmonic acid (JA) treatment was only observed in a root-specific manner consistent with the JA-inducible expressions of DP biosynthetic genes in roots. In reverse genetic approaches on OsTGAP1-overexpressing and OsTGAP1-knockdown plants, expressions of the biosynthetic genes relevant for DP accumulation were found to be remarkably increased and decreased, respectively. Reporter analysis in planta revealed that OsTGAP1 activated the promoters of OsDXS3 and momilactone biosynthetic gene OsKSL4, presumably through binding to the TGACGT motif. Furthermore, cocultivation experiments with barnyard grass suggested that the allelopathic effect of knockdown and overexpression of OsTGAP1 was significantly changed compared with the controls. These results demonstrate that OsTGAP1 positively regulates DP accumulation via the transcriptional regulation of DP biosynthetic genes in rice roots, and this is indispensable for maintaining allelopathic interactions with paddy weeds by regulating the production of specialized metabolites like momilactones., (© 2017 Scandinavian Plant Physiology Society.)

Jasmonic acid (JA) plays central roles in various events in plants, especially defence against pathogens and insects. The basic helix-loop-helix (bHLH) transcription factor MYC2 has attracted attention as a master regulator of JA signalling in dicotyledonous plants. However, how MYC2 functions in monocotyledonous plants, including agriculturally important crops such as cultivated rice, has been poorly understood. To elucidate the comprehensive effects of rice MYC2 (OsMYC2) on the JA-inducible transcriptional modifications, we performed RNA-sequencing by using OsMYC2-knockdown plants (osmyc2RNAi). In osmyc2RNAi, JA-inducible expression of many defence-related genes, for example chitinases and proteinase inhibitors, was compromised. Decrease in JA-dependent activation of the biosynthetic pathways of specialised metabolites, especially defence compounds, was also evident in the osmyc2RNAi line. Furthermore, a substantial change was noted in the expression of distinct types of transcription factors, such as MYB-type factors, likely depicting the importance of OsMYC2 in not only defence responses but also other morphogenetic events. Our findings provide fundamental information to understand the overall functions of MYC2 in JA signalling in monocotyledonous plants, which might yield agricultural benefits., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice., Competing Interests: The authors have declared that no competing interests exist.

Plants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane-related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Rice (Oryza sativa) produces diterpenoid phytoalexins (DPs), momilactones and phytocassanes as major phytoalexins. Accumulation of DPs is induced in rice by blast fungus infection, copper chloride or UV light. Here, we describe a rice transcription factor named diterpenoid phytoalexin factor (DPF), which is a basic helix-loop-helix (bHLH) transcription factor. The gene encoding DPF is expressed mainly in roots and panicles, and is inducible in leaves by blast infection, copper chloride or UV. Expression of all DP biosynthetic genes and accumulation of momilactones and phytocassanes were remarkably increased and decreased in DPF over-expressing and DPF knockdown rice, respectively. These results clearly demonstrated that DPF positively regulates DP accumulation via transcriptional regulation of DP biosynthetic genes, and plays a central role in the biosynthesis of DPs in rice. Furthermore, DPF activated the promoters of COPALYL DIPHOSPHATE SYNTHASE2 (CPS2) and CYTOCHROME P450 MONOOXYGENASE 99A2 (CYP99A2), whose products are implicated in the biosynthesis of phytocassanes and momilactones, respectively. Mutations in the N-boxes in the CPS2 upstream region, to which several animal bHLH transcription factors bind, decreased CPS2 transcription, indicating that DPF positively regulates CPS2 transcription through the N-boxes. In addition, DPF partly regulates CYP99A2 through the N-box. This study demonstrates that DPF acts as a master transcription factor in DP biosynthesis., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Gibberellins (GAs) are diterpenoid phytohormones that regulate various aspects of plant growth. Tetracyclic hydrocarbon ent-kaurene is a biosynthetic intermediate of GAs, and is converted from geranylgeranyl diphosphate, a common precursor of diterpenoids, via ent-copalyl diphosphate (ent-CDP) through successive cyclization reactions catalysed by two distinct diterpene synthases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) has two ent-CDP synthase genes, OsCPS1 and OsCPS2. It has been thought that OsCPS1 participates in GA biosynthesis, while OsCPS2 participates in the biosynthesis of phytoalexins, phytocassanes A-E, and oryzalexins A-F. It has been shown previously that loss-of-function OsCPS1 mutants display a severe dwarf phenotype caused by GA deficiency despite possessing another ent-CDP synthase gene, OsCPS2. Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack. Furthermore, the OsCPS2 transgene under regulation of the OsCPS1 promoter complemented the dwarf phenotype of an OsCPS1 mutant, oscps1-1. The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice-Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.

Phytoalexins are specialised antimicrobial metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are the major diterpenoid phytoalexins in rice and are synthesised from geranylgeranyl diphosphate, which is derived from the methylerythritol phosphate (MEP) pathway. The hyperaccumulation of momilactones and phytocassanes due to the hyperinductive expression of the relevant biosynthetic genes and the MEP pathway gene OsDXS3 in OsTGAP1-overexpressing (OsTGAP1ox) rice cells has previously been shown to be stimulated by the chitin oligosaccharide elicitor. In this study, to clarify the mechanisms of the elicitor-stimulated coordinated hyperinduction of these phytoalexin biosynthetic genes in OsTGAP1ox cells, transcriptome analysis and chromatin immunoprecipitation with next-generation sequencing were performed, resulting in the identification of 122 OsTGAP1 target genes. Transcriptome analysis revealed that nearly all of the momilactone and phytocassane biosynthetic genes, which are clustered on chromosomes 4 and 2, respectively, and the MEP pathway genes were hyperinductively expressed in the elicitor-stimulated OsTGAP1ox cells. Unexpectedly, none of the clustered genes was included among the OsTGAP1 target genes, suggesting that OsTGAP1 did not directly regulate the expression of these biosynthetic genes through binding to each promoter region. Interestingly, however, several OsTGAP1-binding regions were found in the intergenic regions among and near the cluster regions. Concerning the MEP pathway genes, only OsDXS3, which encodes a key enzyme of the MEP pathway, possessed an OsTGAP1-binding region in its upstream region. A subsequent transactivation assay further confirmed the direct regulation of OsDXS3 expression by OsTGAP1, but other MEP pathway genes were not included among the OsTGAP1 target genes. Collectively, these results suggest that OsTGAP1 participates in the enhanced accumulation of diterpenoid phytoalexins, primarily through mechanisms other than the direct transcriptional regulation of the genes involved in the biosynthetic pathway of these phytoalexins.

WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances., (© 2013 Scandinavian Plant Physiology Society.)

OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Bacterial blight is one of the most destructive rice diseases, which caused by Xoo, and results in yield losses, endangering worldwide food security. Diterpenoid phytoalexins, a type of antimicrobials produced in rice, are critical for resistance to fungal and bacterial pathogens. This article reports the characterization of the cytochrome P450 gene Oscyp71Z2, which belongs to the CYP71Z subfamily. Overexpression of Oscyp71Z2 in rice enhanced resistance to Xoo at the booting stage. The accumulation of phytoalexins was rapidly and strongly induced in Oscyp71Z2-overexpressing plants, and the transcript levels of genes related to the phytoalexin biosynthesis pathway were elevated. The H₂O₂ concentration in Oscyp71Z2-overexpressing plants was reduced in accordance with the increase in ROS-scavenging ability due to the induction of SOD as well as POD and CAT activation. We also showed that suppression of Oscyp71Z2 had no significantly effect on disease resistance to Xoo in rice. These results demonstrated that Oscyp71Z2 plays an important role in bacterial blight resistance by regulating the diterpenoid phytoalexin biosynthesis and H₂O₂ generation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP-OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein-fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels.

The plant hormone jasmonic acid (JA) regulates various developmental processes and plant defence responses to environmental stresses. We previously reported that RERJ1, a JA-inducible transcription factor in rice, is up-regulated by exposure to wounding and drought stress. Here, we demonstrated that the expression of RERJ1 after wounding is regulated in a JA-dependent manner in rice, based on histochemical analysis of RERJ1 promoter-GUS transgenic plants. RERJ1 expression was induced only at the region of injury after wounding, whereas expression was induced in the entire leaf after drought. According to JA measurements of stressed leaves, high accumulation of endogenous JA was only detected around the wound site in a rice leaves, whereas the drought treatment led to uniform accumulation of JA in the entire leaf, suggesting that RERJ1 will be a useful marker gene for studies on localization of JA in rice. Nuclear localization and transactivation ability of RERJ1 were also demonstrated. These results suggest that RERJ1 plays a role as a transcriptional activator for regulating stress-inducible gene expression, with a strong correlation to JA accumulation in the stressed region.

We performed extensive functional characterization of diterpenoid phytoalexin biosynthetic genes in rice, and found that the genes for the biosynthesis of the major diterpenoid phytoalexins, phytocassanes and momilactones, are clustered on chromosomes 2 and 4, and that their expression is coordinately induced in rice cells after elicitation. Isopentenyl diphosphate, an early precursor of diterpenoid phytoalexins, was found to be synthesized through the plastidic methylerythritol phosphate pathway. We also found that chitin elicitor receptor kinase OsCERK1 and a mitogen-activated protein kinase cascade, the OsMKK4-OsMPK6 cascade, play essential roles in the elicitor-induced production of diterpenoid phytoalexins. In addition, a basic leucine zipper transcription factor, OsTGAP1, was identified as a key regulator of the coordinated expression of the clustered genes and the methylerythritol phosphate pathway genes. Naringenin 7-O-methyltransferase (OsNOMT) was also identified as a key enzyme in the biosynthesis of another major rice phytoalexin, sakuranetin.

Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with a great impact on stress-induced sakuranetin production.

Sakuranetin, the major flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl 2 or jasmonic acid (JA), or phytopathogenic infection. In addition to sakuranetin's biological significance on disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin, a precursor of sakuranetin, by naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified protein, termed OsCOMT1, exhibited caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1 T-DNA tagged mutant, we determined that OsCOMT1 did not contribute to sakuranetin production in rice in vivo. Therefore, we took advantage of the oscomt1 mutant to purify OsNOMT. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps resulting in a 400-fold purification from the crude enzyme preparation with a minor band at an apparent molecular mass of 40 kDa in the purest enzyme preparation. Matrix-assisted laser desorption/ionization time of flight/time of flight analysis showed that the 40 kDa protein band included two O-methyltransferase-like proteins, but one of the proteins encoded by Os12g0240900 exhibited clear NOMT activity; thus, this gene was designated OsNOMT. Gene expression was induced by treatment with jasmonic acid in rice leaves prior to sakuranetin accumulation, and the recombinant protein showed reasonable kinetic properties to NOMT. Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms. This finding also allows for the generation of disease-resistant and sakuranetin biofortified rice in the future.

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCF(SLF) in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.

Bud endodormancy in woody plants plays an important role in their perennial growth cycles. We previously identified a MADS box gene, DORMANCY-ASSOCIATED MADS box6 (PmDAM6), expressed in the endodormant lateral buds of Japanese apricot (Prunus mume), as a candidate for the dormancy-controlling gene. In this study, we demonstrate the growth inhibitory functions of PmDAM6 by overexpressing it in transgenic poplar (Populus tremula × Populus tremuloides). Transgenic poplar plants constitutively expressing PmDAM6 showed growth cessation and terminal bud set under environmental conditions in which control transformants continued shoot tip growth, suggesting the growth inhibitory functions of PmDAM6. In the Japanese apricot genome, we identified six tandemly arrayed PmDAM genes (PmDAM1-PmDAM6) that conserve an amphiphilic repression motif, known to act as a repression domain, at the carboxyl-terminal end, suggesting that they all may act as transcriptional repressors. Seasonal expression analysis and cold treatment in autumn indicated that all PmDAMs were repressed during prolonged cold exposure and maintained at low levels until endodormancy release. Furthermore, PmDAM4 to PmDAM6 responses to a short period of cold exposure appeared to vary between low- and high-chill genotypes. In the high-chill genotype, a short period of cold exposure slightly increased PmDAM4 to PmDAM6 expression, while in the low-chill genotype, the same treatment repressed PmDAM4 to PmDAM6 expression. Furthermore, PmDAM4 to PmDAM6 expression was negatively correlated with endodormancy release. We here discuss the genotype-dependent seasonal expression patterns of PmDAMs in relation to their involvement in endodormancy and variation in chilling requirements.

The present study investigated the expressional regulation of PpDAM5 and PpDAM6, two of the six peach (Prunus persica) dormancy-associated MADS-box genes, in relation to lateral bud endodormancy. PpDAM5 and PpDAM6 were originally identified as homologues of Arabidopsis SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 identified in the EVERGROWING locus of peach. Furthermore, PpDAM5 and PpDAM6 have recently been suggested to be involved in terminal bud dormancy. In this study, seasonal expression analyses using leaves, stems, and lateral buds of high-chill and low-chill peaches in field conditions indicated that both genes were up-regulated during the endodormancy period and down-regulated with endodormancy release. Controlled environment experiments showed that the expression of both PpDAM5 and PpDAM6 were up-regulated by ambient cool temperatures in autumn, while they were down-regulated by the prolonged period of cold temperatures in winter. A negative correlation between expression levels of PpDAM5 and PpDAM6 and bud burst percentage was found in the prolonged cold temperature treatment. Application of the dormancy-breaking reagent cyanamide to endo/ecodormant lateral buds induced early bud break and down-regulation of PpDAM5 and PpDAM6 expression at the same time. These results collectively suggest that PpDAM5 and PpDAM6 may function in the chilling requirement of peach lateral buds through growth-inhibiting functions for bud break.

Rice blast is one of the most widespread and destructive plant diseases worldwide. Breeders have used disease resistance (R) genes that mediate fungal race-specific 'gene-for-gene' resistance to manage rice blast, but the resistance is prone to breakdown due to high pathogenic variability of blast fungus. Panicle blast 1 (Pb1) is a blast-resistance gene derived from the indica cultivar 'Modan'. Pb1-mediated resistance, which is characterized by durability of resistance and adult/panicle blast resistance, has been introduced into elite varieties for commercial cultivation. We isolated the Pb1 gene by map-based cloning. It encoded a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. The Pb1 protein sequence differed from previously reported R-proteins, particularly in the NBS domain, in which the P-loop was apparently absent and some other motifs were degenerated. Pb1 was located within one of tandemly repeated 60-kb units, which presumably arose through local genome duplication. Pb1 transcript levels increased during the development of Pb1+ cultivars; this expression pattern accounts for their adult/panicle resistance. Promoter:GUS analysis indicated that genome duplication played a crucial role in the generation of Pb1 by placing a promoter sequence upstream of its coding sequence, thereby conferring a Pb1-characteristic expression pattern to a transcriptionally inactive 'sleeping' resistance gene. We discuss possible determinants for the durability of Pb1-mediated blast resistance., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4(DD) ) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4(DD) also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4(DD) -induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4-OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Although cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca(2+) and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca(2+) sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn(2+)-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.

Production of major diterpenoid phytoalexins, momilactones and phytocassanes, is induced in rice upon recognition of pathogenic invasion as plant defense-related compounds. We recently showed that biosynthetic genes for momilactones are clustered on rice chromosome 4 and co-expressed after elicitation, mimicking pathogen attack. Because genes for most metabolic pathways in plants are not organized in gene clusters, examination of the mechanism(s) regulating the expression of such clustered genes is needed. Here, we report a chitin oligosaccharide elicitor-inducible basic leucine zipper transcription factor, OsTGAP1, which is essential for momilactone biosynthesis and regulates the expression of the five genes in the cluster. The knock-out mutant for OsTGAP1 had almost no expression of the five clustered genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) or production of momilactones upon elicitor treatment. Inductive expression of OsKSL7 for phytocassane biosynthesis was also largely affected in the ostgap1 mutant, although phytocassane accumulation still occurred. Conversely, OsTGAP1-overexpressing lines exhibited enhanced expression of the clustered genes and hyperaccumulation of momilactones in response to the elicitor. Furthermore, enhanced expression of OsKSL7 and hyperaccumulation of phytocassanes was also observed. We also found that OsTGAP1 overexpression can influence transcriptional up-regulation of OsDXS3 in the methylerythritol phosphate pathway, eventually leading to inductive production of diterpenoid phytoalexins. These results indicate that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in inductive diterpenoid phytoalexin production in rice and mainly exerts an essential role on expression of the clustered genes for momilactone biosynthesis.

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross 'Vivot' (S(23)S(fa)) x 'Blanquerna' (S(8)S(fi)) was studied because both cultivars share the same S(f) allele but have a different phenotypic expression: active (S(fa)) in 'Vivot' and inactive (S(fi)) in 'Blanquerna'. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S(fi) pollen from 'Blanquerna' was not able to grow down the pistils of 'Vivot' harbouring the S(fa) allele, confirming the active function of this allele against the inactive form of the same allele, S(fi). As self-compatibility was observed in some S(8)S(23) and S(8)S(fa) individuals of this progeny, the S(f) haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.

OsWRKY53, a chitin oligosaccharide elicitor-responsive rice WRKY gene, has been found to be involved in defense responses in rice. We identified three tandem W-box elements, putative recognition sites for WRKY transcription factors, as cis elements that are essential to the elicitor-responsiveness of OsWRKY53 by deletion and mutation analysis of the promoter by dual luciferase assay.

In vitro assays using recombinant enzymes enabled three cDNAs encoding ent-copalyl diphosphate synthases to be identified in wheat (Triticum aestivum): TaCPS1, TaCPS2, and TaCPS3. The phylogenetic tree and expression analyses suggest that TaCPS3 is responsible for gibberellin biosynthesis, while TaCPS1 and TaCPS2 are possible functional homologs of diterpene cyclase genes OsCPS2 and OsCPS4 involved in phytoalexin biosynthesis in rice.

The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.

Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect.

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.

We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.

This study demonstrates that self-compatible (SC) peach has mutant versions of S haplotypes that are present in self-incompatible (SI) Prunus species. All three peach S haplotypes, S (1), S (2), and S (2m), found in this study encode mutated pollen determinants, SFB, while only S (2m) has a mutation that affects the function of the pistil determinant S-RNase. A cysteine residue in the C5 domain of the S (2m)-RNase is substituted by a tyrosine residue, thereby reducing RNase stability. The peach SFB mutations are similar to the SFB mutations found in SC haplotypes of sweet cherry (P. avium) and Japanese apricot (P. mume). SFB (1) of the S (1) haplotype, a mutant version of almond (P. dulcis) S (k) haplotype, encodes truncated SFB due to a 155 bp insertion. SFB (2) of the S (2) and S (2m) haplotypes, both of which are mutant versions of the S (a) haplotype in Japanese plum (P. salicina), encodes a truncated SFB due to a 5 bp insertion. Thus, regardless of the functionality of the pistil determinant, all three peach S haplotypes are SC haplotypes. Our finding that peach has mutant versions of S haplotypes that function in almond and Japanese plum, which are phylogenetically close and remote species, respectively, to peach in the subfamily Prunoideae of the Roasaceae, provides insight into the SC/SI evolution in Prunus. We discuss the significance of SC pollen part mutation in peach with special reference to possible differences in the SI mechanisms between Prunus and Solanaceae.

In this study, we investigated seasonal changes in protein profiles in dormant flower buds of Japanese apricot (Prunus mume Siebold Zucc.) cultivars 'Ellching', from subtropical Taiwan, and 'Nanko', from temperate Japan. One protein, isolated by two-dimensional polyacrylamide gel electrophoresis of flower bud extracts, was shown by peptide sequencing to be a dehydrin (the group of D-11 LEA (late embryogenesis-abundant) proteins). Patterns of dehydrin protein and transcript accumulation differed between the cultivars, with greater accumulations and longer persistence in 'Nanko' than in 'Ellching'. These differences correspond with the greater requirement for chilling to break flower bud dormancy in 'Nanko' than in 'Ellching'. Our study supports the findings of earlier work comparing dehydrin expression in the bark tissue of the evergreen and deciduous peach (Prunus persica (L.) Batsch) genotypes, and suggests that the role of dehydrin during the dormant season is common to all Prunus species.

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.

Many developmental processes and induced plant responses have been identified that are directly or indirectly influenced by wall-localized, or apoplastic, molecular interactions and signalling pathways. The yeast-based signal sequence trap (YSST) is a potentially valuable experimental tool to characterize the proteome of the wall and apoplast, or 'secretome', although few studies have been performed with plants and to date this strategy has not been coupled with a subsequent analysis to confirm extracellular localization of candidate proteins in planta. This current report describes the use of the YSST, together with transient expression of a selection of identified proteins as fusions with the reporter GFP, focusing on the complex extracellular interactions between peach (Prunus persica) pollen and pistil tissues. The coupled YSST and GFP localization assay was also used to confirm the extracellular localization of a recently identified pistil-specific basic RNase protein (PA1), as has been observed with S-RNases that are involved in self-incompatibility. This pilot YSST screen of pollinated and unpollinated pistil cDNAs revealed a diverse set of predicted cell wall-localized or plasma membrane-bound proteins, several of which have not previously been described. Transient GFP-fusion assays and RNA gel blot analyses were used to confirm their subcellular localization and to provide further insights into their expression or regulation, respectively. These results demonstrated that the YSST strategy represents an effective means either to confirm the extracellular localization of a specific candidate secreted protein, as demonstrated here with PA1, or to conduct a screen for new extracellular proteins.

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.

Rice (Oryza sativa L.) produces ent-copalyl diphosphate (ent-CDP) and syn-CDP as precursors for several classes of phytoalexins and the phytohormones, gibberellins (GAs). It has recently been shown that a loss-of-function mutation of OsCPS1, a gene encoding a putative ent-CDP synthase, results in a severely GA-deficient dwarf phenotype in rice. To clarify the biological functions of the ent- and syn-CDP synthases involved in the biosynthesis of phytoalexins and/or GAs, we isolated two cDNAs, OsCyc1 and OsCyc2, encoding putative diterpene cyclases from ultraviolet (UV)-irradiated rice leaves (cv. Nipponbare). The production of phytoalexins in rice leaves is known to be highly induced by UV treatment. Using a bacterial expression system, we demonstrated that OsCyc1 encodes syn-CDP synthase and that OsCyc2 and OsCPS1 encode ent-CDP synthase. The level of expression of the OsCyc1 and OsCyc2 transcripts in rice leaves increased drastically in response to UV treatment, whereas expression of the OsCPS1 transcript was not induced by UV light. These results suggest that OsCyc1, OsCyc2 and OsCPS1 are responsible for the biosynthesis of momilactones A and B and oryzalexin S, oryzalexins A-F and phytocassanes A-E, and GAs, respectively. Our results strongly suggest the presence of two ent-CDP synthase isoforms in rice, one that participates in the biosynthesis of GAs and a second that is involved in the biosynthesis of phytoalexins.

Many Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). The specificity of this outcrossing mechanism is determined by a minimum of two genes that are located in a multigene complex, termed the S locus, which controls the pistil and pollen specificities. SFB, a gene located in the S locus region, encodes an F-box protein that has appropriate S haplotype-specific variation to be the pollen determinant in the self-incompatibility reaction. This study characterizes SFBs of two self-compatible (SC) haplotypes, S(4') and S(f), of Prunus. S(4') of sweet cherry is a pollen-part mutant (PPM) that was produced by X-ray irradiation, while S(f) of Japanese apricot is a naturally occurring SC haplotype that is considered to be a PPM. DNA sequence analysis revealed defects in both SFB(4') and SFB(f). A 4 bp deletion upstream from the HVa coding region of SFB(4') causes a frame-shift that produces transcripts of a defective SFB lacking the two hypervariable regions, HVa and HVb. Similarly, the presence of a 6.8 kbp insertion in the middle of the SFB(f) coding region leads to transcripts for a defective SFB lacking the C-terminal half that contains HVa and HVb. As all reported SFBs of functional S haplotypes encode intact SFB, the fact that the partial loss-of-function mutations in SFB are present in SC mutant haplotypes of Prunus provides additional evidence that SFB is the pollen S gene in GSI in Prunus.

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.

Japanese apricot ( Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene ( SFB), a candidate gene for pollen- S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5'- and 3'-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB(1) and Pm-SFB(7) of 'Nanko ( S(1) S(7))'. As in the case of SFB of other Prunus species, Pm-SFB(1) and Pm-SFB(7) showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S(1)- to S(8)-haplotypes as well as a self-compatible S(f)-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.

This study characterizes the S6m-haplotype, a mutated S6-haplotype with an altered HindIII cut site, of sour cherry (Prunus cerasus). Inheritance and pollination studies of S-haplotypes from reciprocal crosses between 'Erdi Botermo' (EB; S4S6mSa) and 'Rheinische Schattenmorelle' (RS; S6SaSbSc) revealed that the S6m-haplotype conferred unilateral incompatibility with a non-functional pistil component and a functional pollen component. Expression analyses of S6-RNase and SFB6, a candidate gene for pollen-S, in the S6m-haplotype showed that SFB6 was transcribed in EB pollen, but S6-RNase was not transcribed in EB styles. These results were consistent with data from the inheritance and pollination studies. Inverse PCR for the flanking regions of S6-RNase in the S6- and S6m-haplotypes revealed an approximately 2600 bp insertion present at approximately 800 bp upstream of the S6-RNase in the S6m-haplotype, which is responsible for the alternation of the HindIII cut site and a possible cause of inhibition of the transcription of S6-RNase. SFB6 was present downstream of S6-RNase in both the S6- and S6m-haplotypes and expressed in the same way, supporting the idea that SFB is a good candidate for pollen-S in Prunus.

Stylar proteins of 13 almond (Prunus dulcis) cultivars with known S-genotypes were surveyed by IEF and 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-RNases associated with gametophytic self-incompatibility (SI) in this plant species. RNase activities corresponding to Sa and Sb, two of the four S-alleles tested, were identified by IEF and RNase activity staining. The Sa-RNase band reacted with the anti-S4-serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the Sb-RNase band. When the Sa-RNase band was excised from an IEF gel stained for RNase activity, subjected to SDS-PAGE, and detected by immunoblotting, it appeared that this band consisted of a single protein that reacted with the anti-S4-serum with M(r) of about 28 kDa. With 2D-PAGE and silver staining of the stylar extracts, all four S-proteins could be successfully distinguished from each other in the highly basic zone of the gel. Although Sb-, Sc-, and Sd-proteins had roughly the same M(r) of about 30 kDa, the Sc-protein seemed to be slightly smaller than the Sb-protein and slightly larger than the Sd-protein. In 2D-PAGE profiles as well, the Sa-protein had M(r) of about 28 kDa, apparently smaller than the other three proteins. A bud sport, in which one of the two S-alleles of the original cultivar is impaired, was visualized as a loss of Sc-protein, which is consistent with the previous pollination study. All four S-proteins reacted with the anti-S4-serum, probably because of the differing conformations of these S-proteins in the IEF and 2D-PAGE gels. The Sa-protein in 2D-PAGE appeared to be identical to Sa-RNase in IEF; both had the same M(r) and were reactive with the anti-S4-serum. N-terminal amino acid sequence analysis of the four S-proteins revealed that they were highly homologous to each other and similar to the S-RNases of Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together, RNases in the style are strongly suggested to be associated with the gametophytic SI of almond. This is the first report identifying and characterizing S-RNase in almond.

A cDNA clone corresponding to a gibberellin-responsive gene (CRG16) was isolated from cucumber hypocotyls. CRG16 was deduced to encode an extremely hydrophobic protein of 65 amino acids. The deduced sequence exhibited no significant homology to other proteins. Levels of CRG16 mRNA reflected the gibberellin-induced elongation of cucumber hypocotyls.