Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

基因芯片分析杀雄剂SQ-1诱导小麦生理型雄性不育的基因差异表达

为揭示小麦生理型雄性不育的分子机理,更好地为小麦杂种优势利用提供理论依据和技术支撑,本研究以SQ-1诱导的西农558生理型雄性不育的花药为试验材料,以未经SQ-1处理的西农558的花药为对照,利用基因芯片技术对两者的基因表达差异进行了分析,并对部分基因运用半定量PCR技术进行了验证.结果表明,在55 052个转录本中, 两材料间差异表达的转录本有2 052条, 其中1 294个基因表达上调,758个基因表达下调.功能分类表明这些基因主要参与了毒性物质响应、逆境响应、多糖代谢及信号转导等重要生命过程.为验证芯片数据的可信性, 利用cDNA半定量 PCR 法对11个差异表达显著的基因(Ta.116, Ta.5629, TaAffx.122333, Ta.30726, Ta.13682, Ta.4057, Ta.4101, Ta.4139, Ta.11957, Ta.25934, Ta.27552) 进行验证.结果证明,无论是上调表达的还是下调表达的基因,其表达模式都与基因芯片的检测结果的一致.这些基因可作为育性相关候选基因开展下一步研究.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

小麦Ven型胞质雄性不育植株与可育植株花药蛋白质组差异研究

普通小麦具有偏凸山羊草（Ae. ventricosa）细胞质的不育系为Ven型胞质雄性不育系（Ven cytoplasmic male sterility, Ven CMS）,是粘类小麦CMS的一种类型。该研究对小麦Ven型雄性不育系冀5418A及其同型保持系冀5419B的单核期和二核期的花药进行差异蛋白质组学分析,探讨小麦质核互作雄性不育的分子机制。通过双向电泳分离花药蛋白,基质辅助激光解析飞行时间串联质谱（MALDI TOF TOF）对差异表达蛋白进行质谱鉴定,利用生物信息学进行差异表达蛋白鉴定和功能注释分析。结果表明,在分子量19.0～100.0 kD、等电点4～7线性范围内,共检测到约2 000个蛋白点。2个时期共检测到差异蛋白98个,其中两个时期差异表达变化一致的蛋白点56个;数据库搜索获得鉴定的蛋白点41个,其中18个蛋白的表达量在冀5418A 中显著下调,23个在冀5418B 中明显下调。在不育系和可育系中均有参与能量代谢、活性氧代谢、核糖体合成、花粉物质合成的差异蛋白。GO分析预测差异蛋白生物学过程多涉及电子传递和能量代谢、核糖体代谢、活性氧代谢等,细胞组成主要是在膜区域和线粒体,分子功能主要是DNA和RNA结合功能和水解酶等。KEGG分析表明,较多蛋白分布于碳水化合物代谢、活性氧代谢和蛋白组装和折叠途径。推测不育系冀5418A 的雄性不育性除了涉及能量代谢、活性氧清除过程,核糖体蛋白、伴侣蛋白等也有重要作用,雄性不育性可能还与蛋白质加工、物质合成过程的紊乱有关。     

Differential Expression of Proteins in Response to Molybdenum Deficiency in Winter Wheat Leaves Under Low-Temperature Stress

Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress. Of 13 protein spots that were identified, five spots were involved in the light reaction of photosynthesis, five were involved in the dark reaction of photosynthesis, and three were highly involved in RNA binding and protein synthesis. Before the application of cold stress, four differentially expressed proteins between the Mo deficiency (?Mo) vs. Mo application (+Mo) comparison are involved in carbon metabolism and photosynthetic electron transport. After 48 h of cold stress, nine differentially expressed proteins between the ?Mo vs. +Mo comparison are involved in carbon metabolism, photosynthetic electron transport, RNA binding, and protein synthesis. Under ?Mo condition, cold stress induced a more than twofold decrease in the accumulation of six differential proteins including ribulose bisphosphate carboxylase large-chain precursor, phosphoglycerate kinase, cp31BHv, chlorophyll a/b-binding protein, ribulose bisphosphate carboxylase small subunit, and ribosomal protein P1, whereas under +Mo condition cold stress only decreased the expression of RuBisCO large subunit, suggesting that Mo application might contribute to the balance or stability of these proteins especially under low-temperature stress and that Mo deficiency has greater influence on differential protein expression in winter wheat after low-temperature stress. Further investigations showed that Mo deficiency decreased the concentrations of chlorophyll a, chlorophyll b, and carotenoids; the maximum net photosynthetic rate; the apparent quantum yield; and carboxylation efficiency, even before the application of the cold stress, although the decrease rates were greater after 48 h of cold treatment, which is consistent with changes in the expressions of differential proteins in winter wheat under low-temperature stress. These findings provide some new evidence that Mo might be involved in the light and dark reaction of photosynthesis and protein synthesis.     

A proteomic analysis to identify cold acclimation associated proteins in wild wheat (Triticum urartu L.)

To gain a better understanding of cold acclimation process in wheat, we applied a 2-DE based proteomic approach to discover changes in proteome profile of a diploid wild wheat (Triticum urartu L.) during prolonged cold stress treatment. To this end, plants were grown in pots and the growing seedlings (4-leaf stage) were exposed to cold stress. After 4 weeks of cold acclimation (4–6 °C) and subsequent treatment for 12 h at ?2 °C, samples were collected from control and stressed plants and were subjected to proteome pattern analysis. Among approximately 450 reproducible protein spots displayed in each given 2-DE gels, 34 proteins changed significantly in abundance in response to cold stress. Among them, 25 and 9 proteins were up and down-regulated under stress condition, respectively. Analysis by matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry coupled with non-redundant protein database search allowed the identification of 20 cold-induced proteins. Integrated proteomic and database survey resulted in identification of several cold stress related proteins such as pathogenesis related protein, cold regulated protein, cold-responsive LEA/RAB-related COR protein, oxygen-evolving enhancer protein and oxalate oxidase. The presumed functions of the identified proteins were mostly related to cold acclimation, oxidative stress and photosynthesis. The possible implications of differentially accumulated proteins in acquiring systemic tolerance to freezing stress following exposure to prolonged low temperature will be discussed.     

杀雄剂SQ-1诱导小麦生理型雄性不育小花完整叶绿体差异蛋白质组学的鉴定

为了在蛋白质水平揭示小麦雄性不育的分子遗传机制,以小麦经杀雄剂SQ-1诱导的生理型雄性不育系,以及对应正常可育系所构建的等生理差异系为试材,应用差速离心和蔗糖密度梯度离心,首先分离出供试材料小花的完整叶绿体,制备蛋白样品后采用固相IEF/SDS-PAGE双向凝胶电泳技术对完整叶绿体蛋白质进行了分离、银染,得到了重复性较好的双向电泳图谱．PDQuest 2DE软件分析识别出约150个较为清晰的蛋白质点,采用MALDI-TOF鉴定出的6个差异表达蛋白质分别是：PAP-fibrillin、ATRABB1A、底物同源结构域蛋白/Rho GAP结构域蛋白、铜锌超氧化物歧化酶（Cu/Zn-SOD）、R2R3-MYB转录因子及1个假定蛋白质．生物信息学功能分析暗示,这些蛋白直接参与了花药内激素调节、蛋白质转运、蛋白质互作、活性氧积累及花药的发育,表明SQ-1诱导的小麦生理型雄性不育其败育机理可能就与这些生理代谢过程的变异直接相关．     

杀雄剂SQ-1诱导小麦雄性不育花粉粒差异蛋白质组学研究

采用固相pH梯度/SDS-PAGE双向凝胶电泳对经杀雄剂SQ-1处理和未处理的小麦(Triticum aestivum)成熟期花粉总蛋白质进行了分离, 银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 通过PDQuest 2DE图像软件的分析, 在等电点4～7之间可识别350个以上较为清晰的蛋白质点, 其中差异表达明显的蛋白质点数为21个. 将11个差异点采用基质辅助激光解析电离飞行时间质谱进行了肽质量指纹图谱分析, 采用Mascot软件在Swiss-prot数据库查询, 鉴定出了7个蛋白质, 它们分别是液泡转化酶、动力蛋白轻链TCTEX-1、锰超氧化物歧化酶、果糖-1,6-二磷酸醛缩酶、抗坏血酸过氧化物酶、凝集素蛋白激酶和一种未知功能的蛋白. 对已知蛋白的功能进行分析, 推测杀雄剂SQ-1诱导小麦雄性不育可能与能量代谢失衡、淀粉合成受抑制、活性氧积累、细胞凋亡以及花器官发育调节基因作用失控等有关.     

A Draft Map of Rhesus Monkey Tissue Proteome for Biomedical Research

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.     

A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

Comparative proteomic analysis of indica and japonica rice varieties

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93–11 and Nipponbare). In all, 47 proteins that differed significantly between 93–11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93–11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.     

Analysis of Differential Proteins Induced by Forchlorfenuron in Wheat

To identify differential proteins induced by N-(2-chloro-4-pyridy)-N??-phenylurea or forchlorfenuron (CPPU), two-dimensional electrophoresis was used to study the variations in protein expression of wheat seedlings induced with CPPU. The expressed protein spots were identified by matrix-assisted laser desorption/ionization?Ctime of flight mass spectrometry and protein data bank query. About 800 spots were reproducibly detected in three replicates of each sample. A total of 10 differential proteins were identified, including tRNA delta (2)-isopentenylpyrophosphate transferase (S1), probable peptide ABC transporter ATP-binding protein y4tS (S2), ribosomal RNA large subunit methyltransferase N (S3), microtubule-associated protein 6 (S4), actin-66 (Fragment) (S5), mitochondrial ATP synthase subunit alpha (S6), S-adenosylmethionine synthase 1 (S7), flavin-containing monooxygenase YUCCA4 (S8), GTPase obg (S9), and ribosomal RNA small subunit methyltransferase G (S10). These proteins were closely related to phytohormone synthesis, plant stress response, energy metabolism, protein synthesis, cytoskeleton, and transport. All of these processes may play important roles in wheat seedling development. These findings could provide valuable information for the further study of the functions and underlying mechanism of the action of CPPU. Thus, proteomic analysis revealed that CPPU treatment induces variations in many proteins involved in plant growth and development.