A proteomics approach for identifying osmotic-stress-related proteins in rice

Osmotic stress can endanger the survival of plants. To investigate the mechanisms of how plants respond to osmotic stress, rice protein profiles from mannitol-treated plants, were monitored using a proteomics approach. Two-week-old rice seedlings were treated with 400mM mannitol for 48h. After separation of proteins from the basal part of leaf sheaths by two-dimensional polyacrylamide gel electrophoresis, 327 proteins were detected. The levels of 12 proteins increased and the levels of three proteins decreased with increasing concentration or duration, of mannitol treatment. Levels of a heat shock protein and a dnaK-type molecular chaperone were reduced under osmotic, cold, salt and drought stresses, and ABA treatment, whereas a 26S proteasome regulatory subunit was found to be responsive only to osmotic stress. Furthermore, proteins whose accumulation was sensitive to osmotic stress are present in an osmotic-tolerant cultivar. These results indicate that specific proteins expressed in the basal part of rice leaf sheaths show a coordinated response to cope with osmotic stress.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

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.     

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.     

Proteomics analysis reveals multiple regulatory mechanisms in response to selenium in rice

Selenium (Se) shows both beneficial and toxic effects on plant growth. Rice (Oryza sativa L.) seedlings cultivated under lower concentrations of sodium selenite showed enhanced growth, whereas higher concentrations of sodium selenite repressed seedling growth. To acquire detailed regulatory mechanisms underlying these effects, a comparative proteomics study using 2-dimensional gel electrophoresis and MALDI-TOF/TOF MS was performed. By comparison of gel images between Se treatments and control, 66 and 97 differentially expressed proteins were identified in shoot and root, respectively under at least one of the Se treatment concentrations. Gene Ontology and Clustering analysis reveal primary metabolism, photosynthesis and redox homeostasis are the most highly affected biological processes by Se treatments. Lower Se treatments (2 and 6 mg/L sodium selenite) activated antioxidative system, enhanced photosynthesis and primary metabolism. However, higher Se treatment (10 mg/L sodium selenite) damaged photosynthesis apparatus, inhibited photosynthesis and primary metabolism. Protein ubiquitination and phosphorylation may also play important roles in Se response in rice. In conclusion, our study provided novel insights into Se response in rice at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in higher plants and for producing Se enriched rice cultivars in the future.     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

不同生态区烟草叶片蛋白质组学的比较

为探讨不同生态区烟叶香气风格形成的机理,应用蛋白质双向电泳联用质谱技术,对河南平顶山（浓香型烟叶的典型生态区）和福建龙岩（清香型烟叶的典型生态区）的烟草（Nicotina tobaccum L．cv．K326）叶片蛋白质组成进行了比较研究。结果发现,51个蛋白质在两个生态区发生了差异表达,其中在河南表达量上升的有15个,在福建表达量上升的有25个。另外,还分别有2个和9个蛋白点为在河南和在福建样品中特异表达。采用MALDI-TOF/MS进行肽质量指纹图谱分析,经MSDB、NCBInr和SwissPort数据库查询,共鉴定出25种蛋白质,其中参与叶绿体发育、色素代谢、光合作用相关的蛋白在福建烟区高表达,与糖酵解途径相关的蛋白质在河南烟区中高表达。另外,在两生态区烟叶中都发现有相当数量的特异表达的抗逆和防御蛋白。首次在蛋白质组学水平对不同香气风格烟叶的形成机理进行了探讨。     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

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.     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

干旱胁迫条件下马铃薯耐旱品种宁蒗182叶片蛋白质组学分析

开展马铃薯抗旱分子机理的研究对培育马铃薯抗旱品种, 减少干旱造成的损失至关重要。文章利用双向电泳技术对云南地方耐旱马铃薯品种宁蒗182在干旱与正常处理条件下叶片表达差异蛋白质组进行对比研究。经电泳图谱分析和MALDI-TOF-TOF/MS质谱鉴定获得12个表达差异蛋白点, 并进行了功能分类。结果发现, 在差异蛋白中具有保护马铃薯光和系统以及线粒体正常运转的酶类; 调节该植株对环境胁迫响应的信号传导以及调控其组织内N、C运输系统的功能蛋白, 这些蛋白在受到干旱胁迫时表达量均升高。这一结果揭示出该类蛋白是马铃薯在干旱条件下产生的耐受相关蛋白。文章为阐释马铃薯抗旱品种通过多种路径和水平的调控提高其抗性的分子机理提供了理论依据。     

Molecular pathology of breast apocrine carcinomas: a protein expression signature specific for benign apocrine metaplasia

Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers [15-hydroxyprostaglandin dehydrogenase (15-PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase)] present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15-PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15-PGDH, HMG-CoA reductase, and cyclooxygenase 2 (COX-2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.