N-Acetylchitooligosaccharides,biotic elicitor for phytoalexin production,induce transient membrane depolarization in suspension-cultured rice cells

Summary N-acetylchitooligosaccharides (fragments of chitin) elicit the production of phytoalexin in suspension-cultured rice cells. This oligosaccharide elicitor induced rapid and transient membrane depolarization at sub-nanomolar concentrations. Only the oligomers with a certain degree of polymerization were active, while deacetylated chitooligosaccharides caused no effect. Such specificity coincided well with that for the elicitor activity, suggesting possible involvement of this transient change in membrane potential as one of the initial signals in the signal transduction sequence for the activation of defense responses.     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid     

Resistance to Magnaporthe grisea in transgenic rice with suppressed expression of genes encoding allene oxide cyclase and phytodienoic acid reductase

Linolenic acid (18:3) and its derivative jasmonic acid (JA) are important molecules in disease resistance in many dicotyledonous plants. We have previously used 18:3- and JA-deficient rice (F78Ri) to investigate the roles of fatty acids and their derivatives in resistance to the blast fungus Magnaporthe grisea [A. Yara, T. Yaeno, J.-L. Montillet, M. Hasegawa, S. Seo, K. Kusumi, K. Iba, Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid, Biochem. Biophys. Res. Commun. 370 (2008) 344-347; A. Yara, T. Yaeno, M. Hasegawa, H. Seto, J.-L. Montillet, K. Kusumi, S. Seo, K. Iba, Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of ω-3 fatty acid desaturases, Plant Cell Physiol. 48 (2007) 1263-1274]. However, because F78Ri plants are suppressed in the first step of the JA biosynthetic pathway, we could not confirm the specific contribution of JA to disease resistance. In this paper, we generated two JA-deficient rice lines (AOCRi and OPRRi) with suppressed expression of the genes encoding allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR), which catalyze late steps in the JA biosynthetic pathway. The levels of disease resistance in the AOCRi and OPRRi lines were equal to that in wild-type plants. Our data suggest that resistance to M. grisea is not dependent on JA synthesis.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in theplasma membrane from rice leaf and root cells

Presence of a high-affinity binding protein for N-acetylchitooligosaccharide (fragments of chitin) elicitor in the plasma membrane from rice leaf and root cells was shown by affinity labeling experiments with an ¹²⁵I-labeled N-acetylchitooligosaccharide derivative. Binding studies also showed that binding site in the leaf cells has a high affinity to highly elicitor-active, larger chitin fragments but much lower or no affinity to less elicitor-active or elicitor-inactive oligosaccharides. The amount of the binding protein in the leaf cells was slightly smaller than that in the suspension-cultured cells but much larger compared to that in the root cells. These results indicate the possible- involvement of the elicitor binding protein in the perception of the elicitor signal in intact rice plant.     

Antibacterial diterpenes and their fatty acid conjugates from rice leaves

Six structurally oryzalide-related compounds, oryzadione (1), 2, 3, 4, 5 and 6, were isolated from a neutral fraction of the extract of healthy leaves using a bacterial leaf blight-resistant cultivar of a rice plant, "Norin-27", as a group of antimicrobial substances. Their structures were determined by spectroscopic studies to be kaurane analogues and kaurane analogues conjugated with fatty acids, i.e., 1: ent-15,16-epoxy-kauran-2,3-dione (enol form: ent-15,16-epoxy-2-hydroxy-kauran-1-en-3-one), 2: ent-15,16-epoxy-3beta-hydroxy-kauran-2-one, 3: ent-15,16-epoxy-3-oxa-kauran-2-one, 4: ent-15,16-epoxy-3beta-myristoyloxy-kauran-2-one, 5: ent-15,16-epoxy-3alpha-palmitoyloxy-kauran-2-one, and 6: ent-15,16-epoxy-2beta-palmitoyloxy-kauran-2-one.     

Probing the role of tryptophan-derived secondary metabolism in defense responses against Bipolaris oryzae infection in rice leaves by a suicide substrate of tryptophan decarboxylase

Tryptophan-derived secondary metabolites, including serotonin and its hydroxycinnamic acid amides, markedly accumulate in rice leaves in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by being deposited in cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. In this study, (S)-α-(fluoromethyl)tryptophan (S-αFMT) was utilized to investigate the effects of the inhibition of TDC on the defense responses of rice leaves. S-αFMT, enantiospecifically synthesized from l-tryptophan, effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The inhibition rate increased dependently on the incubation time, indicating that S-αFMT served as a suicide substrate. Treatment of rice seedlings with S-αFMT suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner in B. oryzae-inoculated leaves. The lesions formed on seedlings treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with leaves without S-αFMT treatment. Administrating tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material. In addition, tryptamine administration reduced damage caused by fungal infection. Accordingly, the accumulation of tryptophan-derived secondary metabolites was suggested to be part of the effective defense mechanism of rice.     

Cell-cycle-related variation in proteins in suspension-cultured rice cells

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice (Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which -tubulins and a calreticulin-like protein were identified. The amounts of -tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of -tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the -tubulin proteins during 24 h, suggesting that -tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.     

Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid

Linoleic acid (18:2) and linolenic acid (18:3) are sources for various oxidized metabolites called oxylipins, some of which inhibit growth of fungal pathogens. In a previous study, we found disease resistance to rice blast fungus Magnaporthe grisea enhanced in 18:2-accumulating transgenic rice (F78Ri) in which the conversion from 18:2 to 18:3 was suppressed. Here, we demonstrate that 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibit growth of M. grisea more strongly than their 18:3-derived counterparts. Furthermore, in F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results suggest that the increased accumulation of antifungal oxylipins, such as HPODEs and HODEs, causes the enhancement of disease resistance against M. grisea.     

Phosphorylation-mediated regulation of a rice ABA responsive element binding factor

OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells.     

Influence of boric acid on somatic embryogenesis of a cytosterile line of indica rice

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22 μM to the embryo induction medium containing basal salts, with 9.84 μM 2,4-dichlorophenoxyacetic acid. Boric acid was found to exert different effects on scutellum, coleoptile and root tissues. Increased growth of embryogenic masses with increasing boric acid concentrations was observed on coleoptile tissues of mature seed. Embryogenic response was observed at 161.29 μM boric acid on coleoptile and 241.93 μM boric acid on root tissue. Scutellum tissue showed embryonic response in 161.29 and 241.93 μM boric acid. The results indicate that the boric acid used in standard Murashige and Skoog medium is not sufficient to support induction of somatic embryogenesis in IR-58025 A. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of N-fertilizers,straw, and dry fallow on the nitrogen balance of a flooded soil planted with rice

Summary Nitrogen balance studies were made on rice (Oryza sativa) grown in flooded soil in pots. A low rate of fertilizer (5.64 mg N. kg⁻¹ soil) did not depress the N gain, but a high rate (99.72 mg N. kg⁻¹ soil) elminated the N gain. Soil N loss was negligible since¹⁵N applied as ammonium sulfate and thoroughly mixed with the soil was recovered from the soil-plant system after 3 crops. The observed N gain, therefore, was caused by N₂-fixation, not by a reduction of soil N loss. Straw enhanced N gain at the rate of 2–4 mg per g straw. However, this gain was not observed when soil N availability was high. Dry fallow between rice crops decreased the N gain.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.