Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

Interactive effects of methyl jasmonate and salicylic acid on floret opening in spikelets of Sorghum

Floret opening of excised spikelets in Sorghum bicolor (L.) Moench and Sorghum Sudanesis (Piper) Stapf was significantly stimulated by immersing into 2 mM methyl jasmonate (MeJA) solution. In male sterile (MS) lines of Sorghum bicolor (L.) Moench, floret opening was more sensitive to MeJA than that in maintainer (MT) lines. Salicylic acid (SA) could abolish the effect of MeJA on the opening of spikelets. These data indicate that MeJA plays a role in floret opening in sorghum, and that SA interacts with MeJA in regulating of this process.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

Nitric oxide acts as an antioxidant and delays methyl jasmonate-induced senescence of rice leaves

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by methyl jasmonate (MJ). Senescence of rice leaves was determined by the decrease of protein content. MJ treatment resulted in (1) induction of leaf senescence, (2) increase in H₂O₂ and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (ascorbate peroxidase, glutathione reductase, peroxidase and catalase). All these MJ effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-α-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA+NaNO₂] were effective in reducing MJ-induced leaf senescence. PBN prevented MJ-induced increase in the contents of H₂O₂ and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on MJ-promoted senescence, MJ-increased H₂O₂ content and lipid peroxidation, MJ-decreased GSH and AsA, and MJ-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of MJ-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H₂O₂     

The response of two rice cultivars to external Na/Ca ratio

The response of the rice cultivars ‘M9’ and ‘M-201’ to nutrient cultures salinated at −0.4 MPa with varying ratios of Na and Ca was studied. Although the dry matter production of both cultivars was sensitive to the Na/Ca ratio, this correlation was significant only for M-201. Calcium nutrition was severely affected by the composition of the external solution, and the laminae exhibited Ca-deficiency symptoms at Na/Ca molar ratios of 78 and 198. Sodium concentration in the shoot decreased as the Na/Ca ratio in the external solution decreased. Patterns of Na and Cl distribution in the shoot tissues were similar; both ions were accumulated preferentially in the tillers and older leaves. The Na-induced inhibition of Ca uptake and transport appears to be more limiting to shoot growth of M9 and M-201 than Na toxicity per se.     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.     

水稻茎线虫病研究进展

水稻茎线虫病是水稻主要病害之一,能造成水稻严重减产。在我国水稻茎线虫属于检疫对象。该病害在孟加拉国、印度及缅甸等危害严重。本文结合国内外研究现状,从水稻茎线虫病原、形态、分布、症状等方面综述了水稻茎线虫病的研究进展,以期为我国开展检疫和防治提供参考。     

Submergence effects on rice genotypes during seedling stage: Probing of submergence driven changes of photosystem 2 by chlorophyll a fluorescence induction O-J-I-P transients

The effects of submergence on chlorophyll (Chl) a fluorescence were compared in seven Oryza sativa (L.) cultivars, namely FR 13A, Khoda, Khadara, Kalaputia (tolerant), Sabita, and Hatipanjari (avoiding type), and IR 42 (susceptible). Seedlings were submerged for 4 d under complete darkness. Oxygen concentration of flood water decreased with the period of submergence with concomitant increase in concentration of carbon dioxide. Submergence caused diminution in the amount of total Chl. Genotypic differences were observed for Chl content and survival percentage. Quantification of the Chl a fluorescence transients (JIP-test) revealed large cultivar differences in the response of photosystem 2 (PS2) to submergence. The kinetics of Chl a fluorescence rise showed complex changes in the magnitudes and rise of O-J, J-I, and I-P phases caused by submergence. The selective suppression of the J-I phase of fluorescence especially after 2 d of submergence provided evidence for weakened electron donation from the oxygen evolving complex whereas under severe submergence stress (4 d) both O-J and J-I steps were suppressed greatly with highly suppressed P-step, which resulted in lowering of variable fluorescence. Grouping probability or energetic connectivity between PS2 obtained through JIP-test from the data after 2 d of submergence showed a direct relation with survival percentage, i.e. fluorescence measurements contained the information of the survival chance of a plant under submerged conditions. The information could be used in identifying the submergence tolerant cultivars when the damage is not very severe.     

A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice

The effects of abscisic acid (ABA) and methyl jasmonate (MJ) on growth of rice seedlings were compared. The lowest tested concentration of ABA and MJ that inhibited seedling growth was found to be 4.5 and 0.9 µM, respectively. Growth inhibition by ABA is reversible, whereas that by MJ is irreversible. GA₃ was found to be more effective in reversing inhibition of shoot growth by ABA than by MJ. KCl partially relieved MJ-inhibited, but not ABA-inhibited, growth of rice seedlings. The beneficial effect of K⁺ on growth of rice seedlings in MJ medium could not be replaced by Li⁺, Na⁺ or Cs⁺. MJ treatment caused a marked release of K⁺ into the medium. In order to understand whether cell wall-bound peroxidase activity was inversely related to rice seedling growth, effects of ABA and MJ on cell wall-bound peroxidase activity were also examined. Results indicated that both ABA and MJ increased cell wall-bound peroxidase activity in roots and shoots of rice seedlings. Although MJ (4.5 µM) was less effective in inhibiting root growth than ABA (9 µM), MJ was found to increase more cell wall-bound peroxidase activity in roots than ABA.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Repellency of methyl jasmonate to <Emphasis Type="Italic">Ixodes ricinus</Emphasis> nymphs (Acari: Ixodidae)

In our search for tick repellents of plant origin, to be used as alternatives to commercial arthropod repellents, we investigated the effect of the well known plant signaling compound methyl jasmonate (MJ) using nymphs of the tick Ixodes ricinus. In laboratory tests, pieces of cloth with MJ at 0.075, 0.15, 0.30 and 0.75 mg/cm² yielded increasing repellencies against the nymphs: 57%, 71%, 92% and 99%, respectively, of the nymphs did not cling to the cloth. Repellency of MJ was also investigated in a tick-infested woodland area in central Sweden. Cotton flannel cloths sprayed with 0.05, 0.1 or 0.2 mg/cm² MJ dissolved in acetone were dragged over the ground vegetation. The numbers of nymphs on the treated cloths were significantly lower than those on the untreated cloth. Thus, MJ has, at the concentrations tested, significant repellent activity against I. ricinus nymphs.     

Methyl jasmonate elicits a differential antioxidant response in light- and dark-grown canola (Brassica napus) roots and shoots

Since jasmonates have been shown to mimic some of the plant'sresponses to stress, the effect of methyl jasmonate on antioxidant enzymes andcompounds was investigated in roots and shoots of light- and dark-grown canola(Brassica napus cv. Westar). The pattern of superoxidedismutase isoforms activity was also investigated. When enzyme activities werecalculated on a per gram of fresh weight basis, nearly all enzymes examinedshowed enhanced activity. However, when these activities were calculated basedon the amount of protein, methyl jasmonate induced an increase only insuperoxide dismutase activity in the roots of both light- and dark-grownseedlings. The ascorbate level was found to be higher in treated shoots,whereasthe glutathione level was found to be higher in treated roots. We conclude thatthe plant's antioxidant response to methyl jasmonate may be mainlydetermined by the type of tissue rather than by the light conditions. However,this last factor appeared to be involved in some antioxidant componentresponse,e.g. catalase activity and glutathione content.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

Molecular analyses of the rice tubby-like protein gene family and their response to bacterial infection

Tubby-like protein family has been identified in various multicellular organisms, indicating its fundamental functions in the organisms. However, the roles of plant tubby-like proteins are unknown. In this study, we have defined the tubby-like protein gene (OsTLP) family with 14 members in rice. Most of the OsTLPs harbor a tubby domain in their carboxyl terminus and an F-box domain in the amino terminus. The expression of all the OsTLPs was induced on infection of Xanthomonas oryzae pv. oryzae, which causes bacterial blight, one of the most devastating diseases of rice worldwide. The maximal expression levels were observed at 2–8 h after infection for all the genes. Eight of the 14 OsTLPs were also responsive to wounding. All the OsTLPs showed differential expression in different tissues at different developmental stages. However, four pairs of the 14 OsTLPs, with each pair having high sequence similarity and distributing on the similar position of different chromosomes, showed similar expression pattern in different tissues, indicating their direct relationship in evolution. These results suggest that the OsTLP family is involved in host–pathogen interaction and it may be also associated with other physiological and developmental activities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

The effect of elevated CO2 concentration on leaf chlorophyll and nitrogen contents in rice during post-flowering phases

The effect of elevated CO₂ concentration (CE) on leaf chlorophyll (Chl) and nitrogen (N) contents and photosynthetic rate (P_N) was evaluated during the post-flowering stages of rice grown at CE (570 ± 50 μmol mol⁻¹) in open top chamber (OTC), at ambient CO₂ concentration (∼ 365 μmol mol⁻¹) in OTC and at open field. Thirty-five day old seedlings were transplanted in OTCs or in field and allowed to grow till maturity. Chl and N contents were highest at the time of flowering and thereafter it started to decline. The rate of decline in Chl and N contents was faster in plants grown under CE mostly in later part of growth. Irrespective of treatment difference, flag leaf contained the highest amount of Chl and N than penultimate and third leaf. The higher P_N was observed in leaves under CE than in the leaves in other two growing conditions. Considering growth stage, P_N was the highest at flowering which reduced at the later part of growth due to degradation of Chl and N content of the leaf. Under CE it was 40.02 μmol m⁻² s⁻¹ at flowering and it reduced to only 14.77 μmol m⁻² s⁻¹ at maturity stage. The beneficial effect of CE in increasing leaf P_N may be maintained by applying extra dose of nitrogen at the later stages of plant growth.