Effect of early cold stress on the maturation of rice anthers

Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.     

ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice

Cold temperatures cause pollen sterility and large reductions in grain yield in temperate rice growing regions of the world. Induction of pollen sterility by cold involves a disruption of sugar transport in anthers, caused by the cold-induced repression of the apoplastic sugar transport pathway in the tapetum. Here we demonstrate that the phytohormone ABA is a potential signal for cold-induced pollen sterility (CIPS). Cold treatment of the cold-sensitive cultivar Doongara resulted in increased anther ABA levels. Exogenous ABA treatment at the young microspore stage induced pollen sterility and affected cell wall invertase and monosaccharide transporter gene expression in a way similar to cold treatment. In the cold-tolerant cultivar R31, ABA levels were significantly lower under normal circumstances and remained low after cold treatment. The differences in endogenous ABA levels in Doongara and R31 correlated with differences in expression of the ABA biosynthetic genes encoding zeaxanthin epoxidase (OSZEP1) and 9-cis-epoxycarotenoid dioxygenase (OSNCED2, OSNCED3) in anthers. The expression of three ABA-8-hydroxylase genes (ABA8OX1, 2 and 3) in R31 anthers was higher under control conditions and was regulated differently by cold compared with Doongara. Our results indicate that the cold tolerance phenotype of R31 is correlated with lower endogenous ABA levels and a different regulation of ABA metabolism.     

Characterisation of rice anther proteins expressed at the young microspore stage

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.     

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.     

Two-Dimensional Electrophoretic Analysis of Soluble Leaf Proteins of a Salt-sensitive （Triticum aestivum） and a Salt-tolerant （T. durum） Cultivar in Response to NaCI Stress

In this research, 3-day-old etiolated wheat seedlings of Triticum aestivum L. cv. Ceyhan-99 （salt-sensitive） and T. durum Desf. cv. Firat-93 （salt-tolerant） were grown in control and salt （150 mmol/L NaCl） treatments at a 15/25℃ temperature regime in the light for 12 days. Soluble proteins extracted from the first leaf tissues of two cultivars were analyzed by twodimensional （2-D） electrophoresis in order to detect NaCl-induced changes. The soluble leaf protein profiles of cultivars were observed to be similar. However, quantitative differences in 74 proteins were detected in the salt treatment group, compared to the control. Among the 74 protein spots, 14 were common for two cultivars. As a result of NaCl treatment, two low-molecular-weight （LMW） proteins （28.9 and 30.0 kDa） and one intermediate-molecular-weight （IMW） protein （44.3 kDa） in cv. Ceyhan-99 and six LMW proteins （18.6, 19.4, 25.7, 25.9, 26 and 27.6 kDa） in cv. Firat-93 were newly synthesized. The newly synthesized proteins were specific to each cultivar. In the Firat-93 cultivar, four proteins with LMW （24.8-27.9 kDa） were completely lost in NaCl treatment. Moreover, these four protein spots were not observed in both protein profiles of cv. Ceyhan-99. Most of these proteins were in acidic character （pl 〈6.0-6.9） and low molecular weight （〈31.6 kDa）. It is suggested that the newly synthesized or completely lost LMW proteins may be important for cultivars differing in sensitivity towards NaCl.     

Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.     

温敏核不育水稻花药蛋白质组初步分析

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,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 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和 1种未知功能的蛋白质     

Aluminum triggers broad changes in microRNA expression in rice roots

MicroRNAs are small 21-nucleotide RNA molecules with regulatory roles in development and in response to stress. Expression of some plant miRNAs has been specifically associated with responses to abiotic stresses caused by cold, light, iron, and copper ions. In acid soils, aluminum solubility increases, thereby causing severe damage to plants. Although physiological aspects of aluminum toxicity in plants have been well characterized, the molecular mediators are not fully elucidated. There have been no reports about miRNA responses to aluminum stress. Modulation of miRNA expression may constitute a key element to explain the mechanisms implicated in aluminum toxicity and tolerance. We examined the expression of at least one miRNA member from each miRNA family in rice roots of Oryza sativa spp indica cv. Embrapa Taim and Oryza sativa spp japonica cv. Nipponbare under high concentrations of aluminum. Forty-six miRNA families were effectively detected by quantitative PCR. Among these, 13 were down-regulated and six were up-regulated in roots of the Nipponbare cultivar after 8 h of aluminum treatment. In roots of the Embrapa Taim cultivar, five miRNAs were down-regulated and three were up-regulated. Analyses of their putative targets suggest that these rice miRNAs are involved in the regulation of various metabolic pathways in response to high concentrations of aluminum.     

玉米T型细胞质雄性不育系与相应保持系线粒体蛋白质组中的差异蛋白

利用固相pH3—10梯度双向凝胶电泳．对玉米T型细胞质雄性不育系（T—CMS）及其相应保持系叶片（苗期、拔节期、孕穗期）,胚轴,胚根和花药（花粉母细胞减数分裂期、花粉粒小孢子单-双核期）线粒体蛋白质组中的差异蛋白进行分析。PDQuest2D图像软件分析表明,苗期和拔节期叶片约有150个蛋白质斑点,胚轴和胚根中可识别出150个线粒体蛋白质斑点,花药中约有100个斑点。利用MALDI—TOF—MS方法．运用MASCOT软件于NCBI进行数据查询．对T—CMS与相应保持系中存在的差异蛋白进行归属鉴定,在T—CMS中存在,保持系中缺失的线粒体蛋白质有：r40cl protein（胚轴中）,mature anther—specific protein,DNA—directed RNA polymerase 23kD subunit．hexokinaseⅡ和T—CMS中缺失而在保持系中存在的有：glutathione S—transferase．putative protein。其中T—CMS与相应保持系间．线粒体蛋白质组呈现出差异的组织有胚轴、胚根和小孢子单-双核期的花药。叶片的不同发育时期线粒体蛋白质组呈现明显变化．但T—CMS与保持系间没有差异。在小孢子单-双核期（花粉败育期）的花药中,T—CMS与保持系间线粒体蛋白质组出现明显差异,线粒体蛋白质组出现变异的时期与花粉败育时期相一致。     

Primary style protein expression in the self-incompatible/compatible apricot by the 2D-DIGE technique

In order to explore the molecular mechanism underlying self-incompatibility (SI) in the apricot (Prunus armeniaca L.) at the proteome level, we examined the style proteomes at different stages of flower development: small bud, big bud, 24h after self-pollination and 24h after cross-pollination with cultivar Badanshui in the SI apricot cultivar Xinshiji and the self-compatible (SC) apricot cultivar Katy by 2D fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). About 1500 style protein spots were detected; 66 were expressed differently in the four stages in Xinshiji. About 1600 style protein spots were detected; 143 were expressed differently in the four stages of flower development in Katy. In Xinshiji, one protein was expressed specifically, four proteins showed up-regulated expression and twenty-nine proteins showed down-regulated expression in the cross-pollinated style compared to the self-pollinated style. Thirteen proteins were identified unambiguously. In Katy, three proteins were expressed specifically, five proteins showed up-regulated expression and thirteen proteins showed down-regulated expression in the cross-pollinated style compared to self-pollinated style. Seven proteins were identified unambiguously. The different reactions of the style at the proteomic level were triggered in Xinshiji and Katy by self pollen and non-self pollen.     

滤减UV-B辐射对烤烟蛋白质组变化的影响

为研究不同UV-B辐射强度对烤烟生理代谢及调控途径的影响,应用蛋白质双向电泳联用质谱技术,以云南普遍种植的烤烟K326为试验材料,通过覆盖不同透明薄膜滤减UV-B辐射的方式,对75.8%(聚乙烯膜,处理1)和37.5%(麦拉膜,处理2)UV-B辐射透过率处理下K326的蛋白质组和相关生理性状进行了比较。结果表明:在蛋白质组中有10个蛋白在这两类处理下蛋白差异表达显著;与处理1相比,在处理2的K326叶片中有5个蛋白上调表达,5个蛋白下调表达;通过质谱分析共鉴定出8种功能明确的蛋白质,其中差异表达的10个蛋白中有3个与氧化还原相关,3个与光合作用相关,1个是参与能量代谢的激酶蛋白,1个是RNA结合蛋白,另外还有2个未知功能的蛋白待探明,在蛋白质组水平对不同UV-B辐射强度与烤烟生长发育的关系进行了初步研究;而在K326的生理成熟期、过渡期及工艺成熟期,处理2的净光合速率(Pn)均高于处理1,这与所鉴定出的3个与光合作用有光的蛋白在处理2中上调表达趋势一致;相比之下,处理1的K326发育进程较快,茎粗等形态指标及比叶重均比处理2高。     

Differentially expressed proteins in the interaction of Xanthomonas axonopodis pv. citri with leaf extract of the host plant

The present study reports the expression of proteins of Xanthomonas axonopodis pv. citri in response to different growth conditions. The bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a nonhost plant (passion fruit). The protein profiles were analyzed by two-dimensional gel electrophoresis (2-DE). Twelve differential spots (induced, up- and down-regulated and repressed) were observed in the protein profiles of the bacterium cultivated in citrus extract (susceptible host) when compared to that of MM1. The 2-DE profile of the bacterium cultured in the complex medium nutrient yeast glycerol was also obtained and the comparison with that of MM1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and the putative functions were assigned by homology searches in databases. Two constitutively expressed proteins, B4 and B5, were identified as pseudouridine synthase and elongation factor P, respectively. The large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase and a sulfate binding protein were found as specifically up-regulated in the presence of citrus extracts. Finally, the heat shock protein G was found exclusively in the complex medium and repressed in all other media.     

Proteome approaches to characterize seed storage proteins related to ditelocentric chromosomes in common wheat (Triticum aestivum L.)

Changes in protein composition of wheat endosperm proteome were investigated in 39 ditelocentric chromosome lines of common wheat (Triticum aestivum L.) cv. Chinese Spring. Two-dimensional gel electrophoresis followed by Coomassie Brilliant Blue staining has resolved a total of 105 protein spots in a gel. Quantitative image analysis of protein spots was performed by PDQuest. Variations in protein spots between the euploid and the 39 ditelocentric lines were evaluated by spot number, appearance, disappearance and intensity. A specific spot present in all gels was taken as an internal standard, and the intensity of all other spots was calculated as the ratio of the internal standard. Out of the 1755 major spots detected in 39 ditelocentric lines, 1372 (78%) spots were found variable in different spot parameters: 147 (11%) disappeared, 978 (71%) up-regulated and 247 (18%) down-regulated. Correlation studies in changes in protein intensities among 24 protein spots across the ditelocentric lines were performed. High correlations in changes of protein intensities were observed among the proteins encoded by genes located in the homoeologous arms. Locations of structural genes controlling 26 spots were identified in 10 chromosomal arms. Multiple regulators of the same protein located at various chromosomal arms were also noticed. Identification of structural genes for most of the proteins was found difficult due to multiple regulators encoding the same protein. Two novel subunits (1B(Z,) 1BDz), the structure of which are very similar to the high molecular weight glutenin subunit 12, were identified, and the chromosome arm locations of these subunits were assigned.     

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.     

人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。