Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

Seedling vigour and the early growth of transplanted rice (Oryza sativa)

Previous studies suggest that the positive response of transplanted rice (Oryza sativa L.) to nursery fertiliser application was due to increased seedling vigour or possibly to increased nutrient content. This paper presents results of two glasshouse experiments designed to test the hypothesis that seedling vigour was responsible for the response of transplanted seedlings to nursery treatments. The aim of the present study was to explore the concept of seedling vigour of transplanted rice and to determine what plant attributes conferred vigour on the seedlings. Seedling vigour treatments were established by subjecting seedlings to short-term submergence (0, 1 and 2 days/week) in one experiment and to leaf clipping or root pruning and water stress in another to determine their effect on plant growth after transplanting. Submerging seedlings increased plant height but depressed shoot and root dry matter and root:shoot ratio of the seedling at 28 days after sowing. After transplanting these seedlings, prior submergence depressed shoot dry matter at 40 days. Nursery nutrient application increased plant height, increased root and shoot dry matter, but generally decreased root:shoot ratio. Pruning up to 60% of the roots at transplanting decreased shoot and root dry matter, P concentration in leaves at panicle initiation (PI) and straw dry matter and grain yield at maturity. By contrast, pruning 30% of leaves depressed shoot and root dry matter by 30% at PI, and root dry matter and straw and grain yield by 20% at maturity. The combined effects of leaf clipping and root pruning on shoot, root and straw dry matter were largely additive. It is concluded that the response of rice yield to nursery treatments is largely due to increased seedling vigour and can be effected by a range of nutritional as well as non-nutritional treatments of seedlings that increase seedling dry matter, nutrient content, and nutrient concentration. Impairment of leaf growth and to a lesser extent root growth in the nursery depressed seedling vigour after transplanting. However, rather than increasing stress tolerance, seedling vigour was more beneficial when post transplant growth was not limited by nutrient or water stresses.     

Physiological and proteomic analysis of young rice leaves grown under nitrogen-starvation conditions

Rice grown in anaerobic waterlogged soil accumulates ammonium as a major source of nitrogen (N). We have compared the physiological symptoms of rice seedlings subjected to N-starvation stress with those receiving sufficient N, based on measurements of shoot/root length and weight and an analysis of protein expression patterns. N starvation marginally increased root growth but notably decreased shoot biomass. N uptake was reduced by >50% in the roots and shoots of N-starved seedlings. To better understand the mechanism of N starvation in rice, we performed a comparative proteome analysis of proteins isolated from rice leaves. Twenty-five differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry and electron spray ionization quadrupole TOF. Functional analysis of the N-starvation response proteins suggested their involvement in protein synthesis and fate, metabolism, and defense. These results indicate that these proteins may play important roles in regulating the plant’s complex adaptation responses for N use during N starvation. The proteins may be useful for further characterization of protein function in plant N nutrition.     

Maize seedling morphology and defence hormone profiles,but not herbivory tolerance,were mediated by domestication and modern breeding

We addressed whether Zea seedling morphology relevant to performance, defence hormone profiles and tolerance of a phloem‐feeding, specialist herbivore were affected by two processes, plant domestication and modern breeding. Domestication effects were inferred through comparisons between Balsas teosintes (Zea mays parviglumis) and landrace maizes (Z. mays mays), and modern breeding effects through comparisons between landrace maizes and inbred maize lines. Specifically, we compared seedling forms (a composite measure of leaf length, average stem diameter, shoot wet weight, shoot dry weight, total root length, root wet weight, and root dry weight), shapes (forms scaled by seedling dry weight, a proxy for seedling size), and defence hormone profiles among Balsas teosinte and landrace and inbred line maizes, exposed or unexposed to feeding by Dalbulus maidis. Our results suggested that domestication as well as modern breeding strongly mediated both seedling form and shape. Form was more similar between landrace and inbred maize than between Balsas teosinte and landrace maize, suggesting that domestication affected seedling form more than modern breeding. In contrast, shape was more similar between Balsas teosinte and landrace maize than between landrace and inbred maizes, suggesting that modern breeding affected seedling shape more than domestication. Additionally, seedling shoot : root ratios appeared to have been mediated by domestication, but not by modern breeding. In broad terms, individual seedling structures relevant to seedling ecology in wild or managed environments, such as leaf and root lengths, and shoot and root masses, were enlarged with domestication and reduced with modern breeding. Herbivory did not affect seedling shape, but had a weak effect on form so that seedlings were slightly larger in the absence versus presence of D. maidis. Also, both domestication and modern breeding seem to have mediated seedling hormone profiles, with breeding more strongly mediating profiles than domestication. Jasmonic acid isoleucine (JA‐Ile) and salicylic acid (SA) were induced by herbivory in both teosinte and maize. The hormone profiles assays collectively suggested that domestication and modern breeding altered constitutive levels of SA, abscisic acid and JA‐related (JA‐Ile and oxo‐phytodienoic acid) hormone levels in seedlings, particularly by increasing the levels of SA and decreasing those of JA‐related hormones. Altogether, our results suggested that maize domestication and modern breeding significantly altered seedling form, shape, ecologically relevant morphological traits (e.g. leaf and root lengths, and shoot and root masses) and hormonal defences, but not tolerance of D. maidis herbivory.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Effects of application of Rhodopseudomonas sp. on seed germination and growth of tomato under axenic conditions

Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strains KL9 and BL6 were identified as Rhodopseudomonas spp. by 16S rDNA sequence analysis. Rhodopseudomonas strain KL9 maximally produced 5.56 mM/ min/mg protein and 67.2 microM/min/mg protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. The germination percentage of tomato seed, total length, and dry mass of germinated tomato seedling increased by 30.2%, 71.1%, and 270.8%, respectively, compared with those of the uninoculated control 7 days after inoculation of strain KL9. The lengths of the root and shoot of germinated seedling treated with 3 mM tryptophan, a precursor of IAA, increased by 104.4% and 156.5%, respectively, 7 days after inoculation of strain KL9. Rhodopseudomonas KL9 increased 123.5% and 54% of the root and shoot lengths of germinated seedling, respectively, treated with 15 mM glycine and succinate, precursors of ALA. This plant growth promoting capability of purple nonsulfur bacteria may be a candidate for a biofertilizer in agriculture.     

Chitinase induced by jasmonic acid,methyl jasmonate,ethylene and protein phosphatase inhibitors in rice

Chitinase is a pathogenesis-related protein that hydrolyzes chitin, a major component of fungal cell walls. Two-week-old rice seedling leaf, leaf sheath and root tissues responded to an exogenous treatment by jasmonic acid (JA) with induction of the chitinases as determined by immunoblot analysis using an anti-endochitinase antibody. Induced accumulation of these chitinases was observed within 24 to 48 h in the leaf sheaths, leaves and roots. Besides, ethylene generator ethephon and abiotic stressor copper could also induce chitinases accumulation among various plant hormones and stress agents examined. Cycloheximide effectively blocked their accumulation by JA, suggesting that de novo protein synthesis is required. Partial blockage of the induced accumulation of chitinases by NADPH oxidase inhibitor and free radical scavengers suggested involvement of reactive oxygen species. Moreover, induced accumulation of these chitinases also by methyl jasmonate and certain protein phosphatase inhibitors indicated their potential importance and wider role in rice seedlings.     

Effect of aqueous leaf extract of common lambsquarters (Chenopodium album L.) and NaCl on germination and seedling growth of rice

Aqucous leaf extract of common lambsquarters (Chenopodium album L.) was evaluated alone or in combination with NaCl for its effect on germination and rice seedling growth. The treatments have no effect on germination. However, growth of both shoot and root significantly decreased under NaCl alone and in combination with leaf extract. Root growth was affected more than the shoot and the effect of NaCl was accentuated in the presence of leaf extract.     

Comparison of QTLs for rice seedling morphology under different water supply conditions

The variation of seedling characteristics under different water supply conditions is strongly associated with drought resistance in rice (Oryza sativa L.) and a better elucidation of its genetics is helpful for improving rice drought resistance. Ninety-six doubled-haploid (DH)rice lines of an indica and japonica cross were grown in both flooding and upland conditions and QTLs for morphological traits at seedling stage were examined using 208 restriction fragment length polymorphism (RFLP) and 76 microsatellite (SSR) markers. A total of 32 putative QTLs were associated with the four seedling traits: average of three adventitious root lengths (ARL), shoot height (SH), shoot biomass (SW), and root to shoot dry weight ratio (RSR). Five QTLs detected were the same under control and upland conditions. The ratio between the mean value of the seedling trait under upland and flooding conditions was used for assessing drought tolerance. A total of six QTLs for drought tolerance were detected. Comparative analysis was performed for the QTLs detected in this case and those reported from two other populations with the same upland rice variety Azucena as parent. Several identical QTLs for seedling elongation across the three populations with the positive alleles from the upland rice Azucena were detected, which suggests that the alleles of Azucena might be involved in water stress-accelerated elongation of rice under different genetic backgrounds. Five cell wall-related candidate genes for OsEXP1, OsEXP2, OsEXP4, EXT, and EGase were mapped on the intervals carrying the QTLs for seedling traits.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers

This research was undertaken to identify and map quantitative trait loci (QTLs) associated with five parameters of rice root morphology and to determine if these QTLs are located in the same chromosomal regions as QTLs associated with drought avoidance/tolerance. Root thickness, root:shoot ratio, root dry weight per tiller, deep root dry weight per tiller, and maximum root length were measured in three replicated experiments (runs) of 203 recombinant inbred lines grown in a greenhouse. The lines were from a cross between indica cultivar Co39 andjaponica cultivar Moroberekan. The 203 RI lines were also grown in three replicated field experiments where they were drought-stressed at the seedling, early vegetative, and late-vegetative growth stage and assigned a visual rating based on leaf rolling as to their degree of drought avoidance/tolerance. The QTL analysis of greenhouse and field data was done using single-marker analysis (ANOVA) and interval analysis (Mapmaker QTL). Most QTLs that were identified were associated with root thickness, root/shoot ratio, and root dry weight per tiller, and only a few with deep root weight. None were reliably associated with maximum root depth due to genotype-by-experiment interaction. Root thickness and root dry weight per tiller were the characters found to be the least influenced by environmental differences between greenhouse runs. Correlations of root parameters measured in greenhouse experiments with field drought avoidance/tolerance were significant but not highly predictive. Twelve of the fourteen chromosomal regions containing putative QTLs associated with field drought avoidance/tolerance also contained QTLs associated with root morphology. Thus, selecting for Moroberekan alleles at marker loci associated with the putative root QTLs identified in this study may be an effective strategy for altering the root phenotype of rice towards that commonly associated with drought-resistant cultivars.     

Leaf proteomic analysis in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>, Crantz) during plant development,from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student’s t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC–MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.