Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

Proteomics reveals new salt responsive proteins associated with rice plasma membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Proteomics Reveals New Salt Responsive Proteins Associated with Rice Plasma Membrane

The signaling processes in plants that initiate cellular responses to biotic and abiotic factors are believed to be located in the plasma membrane (PM). A better understanding of the PM proteome response to environmental stresses might lead to new strategies for improving stress-tolerant crops. A sub-cellular proteomics approach was applied to monitor changes in abundance of PM-associated protein in response to salinity, a key abiotic stress affecting rice productivity worldwide. Proteome was extracted from a root plasma-membrane-rich fraction of a rice salt tolerant variety, IR651, grown under saline and normal conditions. Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially expressed in response to salt stress. From these, eight proteins were identified by mass spectrometry analysis. Most of the proteins identified are likely to be PM-associated and are known to be involved in several important mechanisms of plant adaptation to salt stress. These include regulation of PM pumps and channels, membrane structure, oxidative stress defense, signal transduction, protein folding, and the methyl cycle. To investigate the correlation between mRNA and protein level in response to salinity, we performed quantitative Real-Time PCR analysis of three genes that were salt responsive at the protein level, including 1,4-Benzoquinone reductase, a putative remorin and a hypersensitive induced response protein. No concordance was detected between the changes in levels of gene and protein expression. Our results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.     

Identification of elicitor‐responsive proteins in rice leaves by a proteomic approach

Experiments were conducted to identify the differentially expressed proteins in rice (Oryza sativa L.) plants after treatment with the glycoprotein elicitor CSB I, purified from ZC₁₃, a race of the rice blast fungus Magnaporthe grisea. The interactions of two near isogenic lines of rice, C101A51 and CO39, with ZC₁₃ resulted in completely incompatible and compatible types, respectively. Proteins were extracted from rice leaves at 12 and 24 h after treatment with CSB I. Temporal changes in total proteins were examined using 2‐DE. Among more than 900 protein spots reproducibly detected on each gel, 11 were up‐regulated, three were down‐regulated and seven were newly induced during, at a minimum, one time point. Twenty‐one differentially expressed proteins were identified by linear ion trap quadrupole (LTQ)‐MS/MS. The identified proteins were classified into six categories based on their putative function reported: (i) defense proteins (PR‐10a, PR‐5 and putative salt‐induced protein), (ii) signal transduction (nucleoside diphosphate kinase and putative profilin), (iii) ROS (Mn‐SOD, Cu/Zn‐SOD, GST and CAT), (iv) programmed cell death (translationally controlled tumor protein), (v) molecule biosynthesis (putative ribosomal protein S5, putative ribosomal protein L12, putative translational elongation factor Tu and putative chaperonin 21 precursor) and (vi) metabolism (putative fructose‐bisphosphate aldolase class‐I, putative malate dehydrogenase, cytoplasmic malate dehydrogenase, putative acid phosphatase, putative transketolase1 and gamma hydroxybutyrate dehydrogenase‐like protein). All of these proteins (except Cu/Zn‐SOD, putative acid phosphatase and translationally controlled tumor protein) were induced faster and to a higher degree in C101A51 than in CO39. These data suggest that the incompatible rice line may possess a more sensitive recognition system that can identify and react to specific chemical, biological or physical triggers in a more efficient manner, thus eliciting an early and fast defense response.     

Proteomic analysis of salt stress-responsive proteins in rice root

Salt stress is one of the major abiotic stresses in agriculture worldwide. We report here a systematic proteomic approach to investigate the salt stress-responsive proteins in rice (Oryza sativa L. cv. Nipponbare). Three-week-old seedlings were treated with 150 mM NaCl for 24, 48 and 72 h. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis. More than 1100 protein spots were reproducibly detected, including 34 that were up-regulated and 20 down-regulated. Mass spectrometry analysis and database searching helped us to identify 12 spots representing 10 different proteins. Three spots were identified as the same protein, enolase. While four of them were previously confirmed as salt stress-responsive proteins, six are novel ones, i.e. UDP-glucose pyrophosphorylase, cytochrome c oxidase subunit 6b-1, glutamine synthetase root isozyme, putative nascent polypeptide associated complex alpha chain, putative splicing factor-like protein and putative actin-binding protein. These proteins are involved in regulation of carbohydrate, nitrogen and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. This study gives new insights into salt stress response in rice roots and demonstrates the power of the proteomic approach in plant biology studies.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

Physiology and proteome responses of two contrasting rice mutants and their wild type parent under salt stress conditions at the vegetative stage

Salinity is one of the major environmental limiting factors that affects growth and productivity of rice (Oryza sativa L.) worldwide. Rice is among the most sensitive crops to salinity, especially at early vegetative stages. In order to get a better understanding of molecular pathways affected in rice mutants showing contrasting responses to salinity, we exploited the power of 2-DE based proteomics to explore the proteome changes associated with salt stress response. Our physiological observations showed that standard evaluation system (SES) scores, Na⁺ and K⁺ concentrations in shoots and Na⁺/K⁺ ratio were significantly different in contrasting mutants under salt stress condition. Proteomics analysis showed that, out of 854 protein spots which were reproducibly detected, 67 protein spots showed significant responses to salt stress. The tandem mass spectrometry analysis of these significantly differentially accumulated proteins resulted in identification of 34 unique proteins. These proteins are involved in various molecular processes including defense to oxidative stresses, metabolisms, photosynthesis, protein synthesis and processing, signal transduction. Several of the identified proteins were emerged as key participants in salt stress tolerance. The possible implication of salt responsive proteins in plant adaptation to salt stress is discussed.     

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834–840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746–2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.     

Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa

Background and Aims

Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos.

Methods

Rice seeds were germinated in the presence or absence of 200 µm Cu for 6 d, and embryos, including newly formed shoots and radicles, were isolated. After proteins were extracted from the germinating embryos and separated by two-dimensional PAGE, 16 proteins in the 6- to 25-kDa range were identified using MALDI-TOF mass spectrometry.

Key Results and Conclusions

Thirteen of the proteins identified, including metallothionein-like protein, membrane-associated protein-like protein, putative wall-associated protein kinase, pathogenesis-related proteins and the putative small GTP-binding protein Rab2, were up-regulated by Cu stress. Three proteins, a putative small cytochrome P450 (CYP90D2), a putative thioredoxin and a putative GTPase, were down-regulated by Cu stress. As far as is known, this study provides the first proteomic evidence that metallothionein and CYP90D2 are Cu-responsive proteins in plants. These findings may lead to a better understanding of plant molecular responses to toxic metal exposure.Key words: Copper, metallothionein, rice, Oryza sativa, proteomics, seed germination     

Grape berry plasma membrane proteome analysis and its differential expression during ripening

High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1-6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process.     

Proteomic analysis of copper stress responses in the roots of two rice (Oryza sativa L.) varieties differing in Cu tolerance

Background and aims

Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice.

Methods

Rice seedlings were exposed to 8 μM Cu for 3 days, with plants grown in the normal nutrient solution containing 0.32 μM Cu serving as the control. Proteins were extracted from the roots and separated by two-dimensional PAGE. Thirty four proteins were identified using MALDI-TOF mass spectrometry.

Results

Thirty-four protein spots were found to be differently expressed in the Cu-stressed roots in at least one variety of rice, including those involved in antioxidative defense, redox regulation, stress response, sulfur and glutathione (GSH) metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in response to Cu stress in the Cu-tolerant variety B1139 compared with the Cu–sensitive variety B1195.

Conclusion

The majority of the proteins showing differential expression in response to Cu exposure are involved in the redox regulation, and sulfur and GSH metabolism, suggesting that these proteins, together with antioxidant enzymes, play an important role in the detoxification of excess Cu and maintaining cellular homeostasis.