Isoflavonoid phytoalexins from fungus-inoculated leaves of Apios tuberosa

A new phytoalexin (apiocarpin) isolated from the fungus-inoculated leaflets of Apios tuberosa has been identified by chemical and spectroscopic pro     

Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

Phenolic compounds detected in rice blast lesions

Salicylic,p-coumaric, and ferulic acids were detected in the acetone-insoluble cell fraction, presumably protein, extracted from the brown discoloured tissues of rice (Oryza sativa L.) leaves infected withPyricularia oryzae Cav. but not from healthy leaves. It is proposed that these phenolic acids are oxidizedin vivo in blast diseased rice leaves, and forms a protein-quinone complex.     

Molecular cloning and differential expression of an aldehyde dehydrogenase gene in rice leaves in response to infection by blast fungus

Aldehyde dehydrogenase (ALDH) superfamily is a group of enzymes metabolizing endogenous and exogenous aldehydes. Using differential display RT-PCR and cDNA library screening, a full-length aldehyde dehydrogenase cDNA (ALDH7B7) was isolated from rice leaves infected by incompatible race of blast fungus Magnaporthe grisea. The deduced amino acid sequence consists of 509 amino acid residues and shares 74∼81% identity with those of ALDH7Bs from other plants. ALDH7B7 expression was induced by blast fungus infection, ultraviolet, mechanical wound in rice leaves and was not detected in untreated rice organs. This gene has also been found to be inducible after exogenous phytohormones application, such as salicylic acid, methyl ester of jasmonic acid and abscisic acid. The function of ALDH7B7 in the interaction process between blast fungus and rice is discussed.     

Methionine-induced phytoalexin production in rice leaves

The application of methionine on wounded rice leaves induced the production of rice phytoalexins, sakuranetin and momilactone A. This induction resulted from stimulation of phenylalanine ammonia-lyase and naringenin 7-O-methyltransferase activity. Jasmonic acid, ethylene, and active oxygen species are important as signal transducers in disease resistance mechanisms. However, although the endogenous level of jasmonic acid rapidly increased in reaction to wound, methionine treatment could not induced endogenous JA production. Ethylene induced the production of the flavonoid phytoalexin, sakuranetin, but did not induce the production of a terpenoid phytoalexin, momilactone A. On the other hand, a free radical scavenger, Tiron, counteracted the induction of both sakuranetin and momilactone A production in methionine-treated leaves. Active oxygen species may be important in methionine-induced production of phytoalexins.     

Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.     

Modification of fatty acids changes the flavor volatiles in tomato leaves

Expression of the yeast Delta9 desaturase gene in tomato (Lycopersicon esculentum Mill.) resulted in changes in the profiles of fatty acids in tomato leaves. Transgenic leaves displayed a dramatic increase in cis-Delta9 16:1, which only existed in a small quantity in control leaves. Also higher, but not as dramatic, were 18:1 and 16:3 fatty acids. Several fatty acids, viz. 16:0, 18:0, and 18:3 declined in transgenic leaves. Changes in fatty acids were accompanied by changes in certain volatile compounds derived from fatty acids. On a percentage basis, most notable increases (>3-fold) were 1-hydroxy-2-butanone, 1-penten-3-ol, heptanal, 3-hexen-1-ol, 2-octanol,cis-3-hexenal, hexanal and 2-nonenal. Several flavor compounds not known to be biochemically derived from fatty acids, viz. 2-ethyl-furan, 5-ethyl-2-[5H]-furanone, eugenol, and 2-ethylthiophene also showed sharp increases in transgenic leaves.     

Characterization of rice mutants with enhanced susceptibility to rice blast

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the F2 segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the F2 population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 F2 plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.     

Glutamate synthase from rice leaves

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a final specific activity of 35.9 mumoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purified enzyme yielded a single protein band on polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was estimated to be 224,000 daltons by Sepharose 6B gel filtration. Electrophoresis of the dissociated enzyme in sodium dodecyl sulfate-polyacrylamide gel gave a single protein band which corresponds to the subunit molecular weight of 115,000 daltons. Thus, it is concluded that the glutamate synthase is composed of two polypeptidic chains exhibiting the same molecular weight. Spectrophotometric analysis indicated that the enzyme is free of iron-sulfide and flavin. The pH optimum was 7.3. The enzyme had a negative cooperativity (Hill number of 0.70) for glutamine, and its K(m) value increased from 270 to 570 mum at a glutamine concentration higher than 800 mum. K(m) values for alpha-ketoglutarate and ferredoxin were 330 and 5.5 mum, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and alpha-ketoglutarate, respectively. Enzyme activity was not detected with pyridine nucleotides as electron donors. Azaserine and several divalent cations were potent inhibitors. The purified enzyme was stabilized by dithiothreitol.     

Blue light-induced unrolling in rice plant leaves

Blue light-induced unrolling of second leaves in rice plants(Oryza sativa L.) was studied. Light in wavelengths of 400–500nm was most effective for the induction of unrolling, whilethat of 500–800 nm had no influence. This blue light actionon unrolling was observed for both dark and light grown seedlings.Several hours of irradiation was required for the inductionof unrolling at a relatively high intensity. Red light had noinfluence on the blue light action. We concluded that blue lightaction on the unrolling of rice leaves is not mediated by thephytochrome system, but by a high energy blue light reactionwhich differs from the unrolling of wheat and barley leaves. (Received March 3, 1979; )     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

A pharmacological approach to test the diffusible signal activity of reactive oxygen intermediates in elicitor-treated tobacco leaves

The capacity of H(2)O(2), the most stable of the reactive oxygen species (ROI), to diffuse freely across biological membranes and to signal gene expression suggests that H(2)O(2) could function as a short-lived second messenger diffusing from cell to cell. We tested this hypothesis in tobacco plants treated with a glycoprotein elicitor. Applied at 50 nM, it induces H(2)O(2) accumulation and the hypersensitive response restricted to the infiltrated zone 1 tissue. Stimulation of a set of defense responses also occurs in the surrounding zone 2 tissue without diffusion of the elicitor. ROI levels in zone 1 were modulated using N-acetyl-L-cysteine (NAC) as a ROI scavenger and Rose Bengal (RB) as a ROI generator. We found that ROI appeared to act as signalling intermediates in pathways leading to salicylic acid accumulation, to PR1, PR5 and 3-hydroxy-3-methylglutarylCoA reductase expression in glycoprotein-treated zone 1 tissues. Compared to the treatment with the elicitor alone, co-infiltration of the glycoprotein and NAC increased the surface of zone 2 showing PR1 and O-methyltransferase expression. Application of RB had the opposite effect. The data suggest that, in our system, ROI did not act as a cell-to-cell diffusible signal to activate PR protein and O-methyltransferase expression in zone 2.     

Reaction of incompatible isolate of Pyricularia grisea on rice leaves stripped of epicuticular wax reflects blast conduciveness of soils

Upland rice cultivars were evaluated in the greenhouse for susceptibility to the rice blast disease caused by Pyricularia grisea Sacc., on two upland soils from the Philippines previously considered to be “blast conducive” and “blast non-conducive”. Under monocyclic inoculation tests plants grown in conducive soil showed significantly greater lesion development than plants of the same cultivar grown in non-conducive soil: cultivars considered to be susceptible to the isolates used showed increased number of susceptible-type lesions; resistant cultivars showed increased number of hypersensitive resistant-type lesions. A similar effect was observed under polycyclic tests where several generations of the pathogen were allowed to develop on the test plants. Dilution of conducive soil with non-conducive soil resulted in a corresponding reduction of disease severity, although this was most pronounced on resistant cultivars. Removal of leaf epicuticular waxes (LEW) using organic solvents increased the number of resistant-type lesions on resistant cultivars grown in both soils following inoculation. Susceptible plants were not suitable for quantifying the relative blast conduciveness of a soil because of the extreme environmental sensitivity of the bioassay and the tendency of lesions to coalesce. Comparing numbers of resistant-type lesions on leaves of plants stripped of LEW and inoculated with an incompatible P. grisea isolate among plants grown in different soils proved to be a satisfactory means of distinguishing the relative blast conduciveness of soils under controlled conditions. This method was field tested in eastern India and results corroborated farmer assessment of which soils were blast conducive. Using incompatible isolate-cultivar combinations and LEW-free leaves is proposed as a simple bioassay for assessing blast conduciveness of soils and should prove useful in regional characterization of rice blast risk.     

Induced resistance in rice against insects

Vaccinations are the mainstay of western preventive medicine, and they have been used to protect some crops against disease and insect pests. We consider rice as a model for protection using induced resistance since it is one of the most important staple crops and there have been significant new developments in: cross-resistance among rice insects, chemical pathways involved in induced resistance, sequencing the rice genome and expression of genes conferring resistance against rice insect pests. Insect attack has been found to cause lesions that kill planthopper eggs and early stages of gall midges. Damaged plants released volatiles that made them less likely to be chosen by planthoppers and more attractive to parasitoids. Chemical elicitors have been developed for dicotyledonous plants and these can induce resistance in rice, although rice does not fit models developed to explain signalling in dicots. For example, salicylic acid did not increase in rice after infection by pathogens and did not appear to be the mobile signal for induced resistance against pathogens although it was involved in induced responses to phloem-feeding insects. Jasmonic acid acted as a signal in some induced responses to pathogens as well as chewing insects. Many of the genes associated with induced resistance in rice have recently been mapped, and techniques are being developed to incorporate them into the genome of cultivated varieties. Attempts to control insect pests of rice will affect interactions with pathogens, predators and parasites, and other organisms in this agroecosystem.     

Proteomic changes in rice leaves during development of field-grown rice plants

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.     

Aldehyde volatiles emitted in succession from mechanically damaged leaves of poplar cuttings

Plant aldehydes are volatiles necessary to defenses against environmental stress. To explore their emissions in response to wounding, we performed gas chromatography-mass spectrometry (GC-MS) on cuttings from poplar (Populus simonii×P. pyramidalis ‘Opera 8277’) that were mechanically damaged to mimic herbivore attack. We detected 16 aldehydes, including 11 linear saturated aldehydes, 3 linear unsaturated aldehydes, and 2 non-linear aldehydes. Emissions of these aldehydes were clearly enhanced by such treatment, and exhibited a similar pattern of change, i.e., increasing in the first 2 h, then sharply decreasing before rising again at about 12 h. Two release peaks for these aldehydes were observed. Therefore, we propose two pathways for the mediation of aldehyde emissions following damage. The first peak may represent emissions from plant storage pools, whereas the second release peak might result from greater formationde novo through an activated synthesis pathway.