Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 ( spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.     

How phenology influences physiology in deciduous forest spring ephemerals

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μ M . Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7- O -methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)-inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4-dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

防卫基因PAL,LOX和PBZ1在水稻白叶枯病成株抗性中的作用(英文)

为了探讨水稻白叶枯病成株抗性是否与防卫基因相关 ,通过RT PCR研究了几种防卫基因PAL、LOX、PBZ1、Cht 1和PR1a的表达 ,同时也分析了PAL和LOX的酶活性变化 .结果表明 ,苗期PAL受病原菌诱导上调表达 ,而成株期病原菌和伤害均可诱导PAL上调表达 ,且成株期诱导表达强于苗期 .LOX在苗期和成株期都可受病原菌诱导表达 ,但成株期表达强于苗期且更持久 .PBZ1在苗期和成株期均受病原菌和伤害诱导表达 ,但成株期表达较苗期早且更强 .PR1a和Cht1没检测到期望的扩增片段 .PAL和LOX的酶活性分析结果与基因表达分析结果一致 .因此 ,PAL、LOX和PBZ1在苗期和成株期的表达差异可能与水稻白叶枯病成株抗性相关 .     

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL,Cht-1, and PBZ1, but not of Lox in rice

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.     

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.     

磷高效水稻根系对低磷胁迫响应的差异蛋白分析

以IR71331为材料,通过水培试验,采用双向电泳分离不同磷浓度下（低磷浓度为0.5 mg·L^-1,对照为10 mg·L^-1）水稻生长3 d和6 d根系差异蛋白.结果表明：与对照相比,低磷胁迫下共有29个蛋白,其中3 d时间点有17个蛋白上调、11个下调、1个新增,6 d时间点有8个上调、19个下调、1个抑制表达、1个无明显变化 .经鉴定,其中的10个差异表达蛋白可归为信号转导相关蛋白、基因表达相关蛋白、代谢相关蛋白离子转运相关蛋白4个功能类群.信号转导相关蛋白分别为富含甘氨酸RNA结合蛋白和类似参与磷酸盐饥饿反应调控子;基因表达相关蛋白分别为推定的mRNA前体剪接因子SF2和推定的AAA蛋白酶家族FtsH;代谢相关蛋白分别为腺苷酸琥珀酸裂解酶、丝氨酸蛋白酶抑制剂(serpin)、S-腺苷蛋氨酸合成酶（SAM）和类似MYB类转录因子;离子转运相关蛋白分别为阳离子转运ATP酶和肌浆网膜蛋白.这些蛋白分别参与了信号识别、信号调控、mRNA 的剪接、信号传递、蛋白质降解、细胞体内离子转运和平衡等生理过程.其中serpin、SAM和MYB类转录因子是水稻响应低磷胁迫的关键蛋白.水稻根系对低磷胁迫存在着一个复杂的抗逆信号应答和代谢调控网络,其作用机理可以通过差异表达的蛋白质得以体现.     

Cloning and Characterization of a Probenazole-Inducible Gene for an Intracellular Pathogenesis-Related Protein in Rice

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) inducesdisease resistance in rice against rice blast fungus. To investigatethe molecular mechanism of probenazole-induced resistance, weisolated and characterized a cDNA clone of a probenazole-induciblegene in rice, which encoded a protein designated PBZ1. Sequenceanalysis revealed that significant homology at the amino acidlevel exists between the predicted PBZ1 protein and intracellularpathogenesis-related (IPR) proteins. Accumulation of PBZ1 mRNAwas not induced by wounding, but markedly induced by inoculationwith rice blast fungus. In addition, it was induced sooner byinoculation with rice blast fungus. In addition, it was inducedsooner by inoculation with an incompatible race than that witha compatible race. On the other hand, when the accumulationof the PBZ1 mRNA was examined after treatment with probenazole-relatedcompounds, it was not fully correlated with anti-rice blastactivity. However, it was induced after treatement with N-cyanomethyl-2-chloro-isonicotinamide(NCI), which belongs to another group of compounds known toinduce disease resistance. Thus, although the accumulation ofthe PBZ1 mRNA was not fully correlated with anti-rice blastactivity, our findings suggest that the PBZ1 gene has an importantfunction during the disease resistance response in rice. (Received June 19, 1995; Accepted October 13, 1995)     

gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.     

Proteomic Analysis of Rice Plasma Membrane-associated Proteins in Response to Chitooligosaccharide Elicitors

Chitooligomers or chitooligosaccharides （COS） are elicitors that bind to the plasma membrane （PM） and elicit various defense responses. However, the PM-bound proteins involved in elicitor-mediated plant defense responses still remain widely unknown. In order to get more information about PM proteins involved in rice defense responses, we conducted PM proteomic analysis of the rice suspension cells elicited by COS. A total of 14 up- or downregulated protein spots were observed on 2-D gels of PM fractions at 12 h and 24 h after COS incubation. Of them, eight protein spots were successfully identified by MS （mass spectrography） and predicted to be associated to the PM and function in plant defense, including a putative PKN/PRK1 protein kinase, a putative pyruvate kinase isozyme G, a putative zinc finger protein, a putative MAR-binding protein MFP1, and a putative calcium-dependent protein kinase. Interestingly, a COS-induced pM5-like protein was identified for the first time in plants, which is a transmembrane nodal modulator in transforming growth factor-β（TGFβ） signaling in vertebrates. We also identified two members of a rice polyprotein family, which were up-regulated by COS. Our study would provide a starting point for functionality of PM proteins in the rice basal defense.     

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.     

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.     

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.     

红莲型水稻细胞质雄性不育花粉总蛋白质初步比较分析

采用固相pH梯度/SDS-PAGE双向电泳对红莲型细胞质雄性不育水稻的不育系(YTA)和保持系(YTB)二核期花粉总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。用PDQuest2DE软件可识别约1500个蛋白质点,其中差异表达的蛋白质点数为120。将其中15个差异点采用基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption/ionizaton time of flight mass spectrometry,MALDI-TOF-MS)进行了肽质指纹图分析,通过采用Mascot软件对MSDB数据库查询,其中7个蛋白质点得到了鉴定。YTA相对于YTB有部分参与物质和能量代谢的蛋白质缺失或表达量降低,这些蛋白质分别是水稻线粒体H -转运ATPase(H -ATPase)α链、盐诱导型膜联蛋白、线粒体NAD -依赖型苹果酶和磷酸核糖焦磷酸合成酶等。这些蛋白质的表达下调或缺失可能与线粒体提供能量不足而导致的花粉不能正常发育有关。线粒体电压依赖性阴离子通道(VDAC)这一重要蛋白质在YTA中的上调表达有可能与花粉败育过程中细胞的程序性死亡相关。