iTRAQ‐based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies

Male and female poplars (Populus cathayana Rehd.) respond differently to nitrogen (N) and phosphorus (P) deficiencies. In this study, an iTRAQ‐based quantitative proteomic analysis was performed. N and P deficiencies caused 189 and 144 proteins to change in abundance in males and 244 and 464 in females, respectively. Compared to N‐ and P‐deficient males, both N‐ and P‐deficient females showed a wider range of changes in proteins that are involved in amino acid, carbohydrate and protein metabolism, and the sexual differences were significant. When comparing the effects of N‐ and P‐deficiencies, N‐deficient females expressed more changes in proteins that are involved in stress responses and gene expression regulation, while P‐deficient females showed more changes in proteins that are involved in energy and lipid metabolism, stress responses and gene expression regulation. The quantitative RT‐PCR analysis of stress‐related proteins showed that males have a better expression correlation between mRNA and protein levels than do females. This study shows that P. cathayana females are more sensitive and have more rapid metabolic mechanisms when responding to N and P deficiencies than do males, and P deficiency has a wider range of effects on females than does N deficiency.     

植物盐胁迫应答蛋白质组学分析

土壤盐渍化是限制植物生长和分布的关键因素之一,揭示植物盐胁迫应答的分子机理是借助分子生物学手段提高植物耐盐性的基础.近年来,人们利用高通量蛋白质组学技术分析了拟南芥、水稻等19种植物的盐胁迫应答蛋白质表达图谱.从植物类群(盐生植物和甜土植物)、组织器官(根、地上部分/茎、胚根和胚轴、叶片、花序和配子体)、细胞(悬浮培养细胞、愈伤组织细胞和单细胞生物)和亚细胞结构(叶绿体、质膜和质外体)几方面整合分析了植物盐胁迫应答蛋白质组表达模式特征,主要特征包括:(1)盐生植物通过全面调节细胞骨架重塑、离子转运和区隔化、渗透平衡、活性氧(ROS)清除、信号转导、光合作用和能量代谢等信号与代谢网络体系,获得相对较高的抗/耐盐能力;(2)植物地上部分(叶片、茎、配子体)或光合组织细胞(悬浮培养细胞、愈伤组织细胞和单细胞盐藻)通过调节参与光合作用、碳和能量代谢、ROS清除过程蛋白质的表达模式应对盐胁迫环境;(3)植物地下部分(根、胚根)通过调控信号转导和离子转运相关蛋白质感知/传递盐胁迫信号并维持离子平衡;(4)花序中参与渗透调节、转录调控、蛋白质加工和ROS清除的蛋白质在盐胁迫条件下变化显著;(5)叶绿体通过调控参与光合作用、蛋白质加工和周转,以及氧化还原系统平衡等过程应对盐胁迫;(6)质外体中参与细胞壁代谢、胁迫防御和信号转导过程的蛋白质受盐胁迫影响明显;(7)细胞膜中参与维持膜结构稳定、物质/离子运输和信号转导过程的蛋白质对植物盐胁迫应答具有重要作用.这些分析为深入研究植物耐盐的分子机制提供了重要信息.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Comparative proteomics analysis of salt response reveals sex-related photosynthetic inhibition by salinity in Populus cathayana cuttings

Male and female poplar ( Populus cathayana Rehd.) cuttings respond differently to salinity stress. To understand these differences better, comparative morphological, physiological, and proteomics analyses were performed. Treatments with different concentrations of NaCl applied to male and female poplar cuttings for 4 weeks showed that females reacted more negatively at the morphological and physiological levels than did males, visible as shriveled leaves, decreased growth, lowered photosynthetic capacities, and greater Na(+) accumulation. The proteome analysis identified 73 proteins from 82 sexually related salt-responsive spots. They were involved in photosynthesis, protein folding and assembly, synthesis and degradation, carbon, energy and steroid metabolism, plant stress and defense, redox homeostasis, signal transduction, and so forth. The sex-related changes of these proteins were consistent with the different morphological and physiological responses in males and females. In conclusion, the higher salt resistance of male P. cathayana cuttings is related to higher expression and lower degradation of proteins in the photosynthetic apparatus, more effective metabolic mechanism and protective system, and greater capacity of hydrogen peroxide scavenging. This research allows us to further understand the possible different management strategies of cellular activities in male and female Populus when confronted by salt stress.     

Decarbonylated cyclophilin A Cpr1 protein protects <Emphasis Type="BoldItalic">Saccharomyces cerevisiae</Emphasis> KNU5377Y when exposed to stress induced by menadione

Cyclophilins are conserved cis–trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.     

水稻叶绿体蛋白质在生长发育过程中的表达研究

在植物中,叶绿体是负责光合作用的细胞器,对叶绿体内的各种生物过程人们已经积累了很多知识,但对叶绿体蛋白质的表达还所知甚少．为了解水稻叶绿体蛋白质在正常生长发育过程中的表达情况,尝试基于抗体的水稻蛋白质组学策略．选取了10个水稻叶绿体基因,利用表达的蛋白质或合成的抗原决定簇片段制备了抗体,用Western blotting检测了相应蛋白质在5个发育时期的根、茎、叶及穗组织中的表达．发现10个蛋白质均在叶片中表达,在根中不表达．与原初反应相关的叶绿素A/B结合蛋白1和2(CAB1和CAB2)、与电子传递相关的放氧增强蛋白1(OEE1)及与活性氧清除相关的过氧还蛋白过氧化物酶(2-CysP)和硫氧还蛋白(Trx)在茎中表达．而在卡尔文循环中发挥作用的Rubisco活化酶(RCA)、甘油醛-3-磷酸脱氢酶(GAPDH)、果糖二磷酸醛缩酶(FBPA)和景天庚酮糖-1, 7-二磷酸酶(SBPase)蛋白质在茎中不表达．在穗中,这些蛋白质的表达时序不同,CAB2和2-CysP在穗发育的全程表达,CAB1和OEE1在中后期表达,而卡尔文循环中的蛋白质只在中期表达．有意思的是,卡尔文循环中的蛋白质表达模式相似,这一结果从蛋白质表达水平支持它们之间的相互衔接关系．此外,实验还揭示了可能的蛋白质修饰、二聚体及不同的转录本现象．将目标基因的表达谱与转录谱进行比较,发现二者间有一定的平行性,但也有明显的区别．以水稻叶绿体蛋白质为对象,直观并相对定量地揭示了它们的表达模式,为阐释其功能提供了信息,也为基于抗体的水稻蛋白质组学策略提供了一个初步数据．     

Sex‐specific responses of Populus yunnanensis exposed to elevated CO2 and salinity

Populus yunnanensis Dode., a native dioecious woody plant in southwestern China, was employed as a model species to study sex‐specific morphological, physiological and biochemical responses to elevated CO₂ and salinity. To investigate the effects of elevated CO₂, salinity and their combination, the cuttings were exposed to two CO₂ regimes (ambient CO₂ and double ambient CO₂) and two salt treatments in growth chambers. Males exhibited greater downregulation of net photosynthesis rate (A_net) and carboxylation efficiency (CE) than females at elevated CO₂, whereas these sexual differences were lessened under salt stress. On the other hand, salinity induced a higher decrease in A_net and CE, more growth inhibition and leaf Cl^? accumulation and more damage to cell organelles in females than in males, whereas the sexual differences in photosynthesis and growth were lessened at elevated CO₂. Moreover, elevated CO₂ exacerbated membrane lipid peroxidation and organelle damage in females but not in males under salt stress. Our results indicated that: (1) females are more sensitive and suffer from greater negative effects than do males under salt stress, and elevated CO₂ lessens the sexual differences in photosynthesis and growth under salt stress; (2) elevated CO₂ tends to aggravate the negative effects of salinity in females; and (3) sex‐specific reactions under the combination of elevated CO₂ and salinity are distinct from single‐stress responses. Therefore, these results provide evidence for different adaptive responses between plants of different sexes exposed to elevated CO₂ and salinity.     

Comparative physiological,ultrastructural and proteomic analyses reveal sexual differences in the responses of Populus cathayana under drought stress

Drought is a major abiotic stress, limiting the survival and growth of young plants. However, little is known about sex‐dependent responses to drought at the proteome level. In this study, we carried out investigations on comparative proteomics, combined with physiological and organelle structure analyses, in males and females of Populus cathayana Rehd. Three‐month‐old poplar cuttings were treated at 30% of field capacity and at 100% field capacity as a control in a greenhouse for 40 days. Drought greatly inhibited plant growth, damaged the photosynthetic system and destructed the structures of chloroplasts, mitochondria and cellular membranes. However, males suffered less from the adverse effects of drought than did females. Using 2‐DE, 563 spots were detected, of which 64 spots displayed significant drought effect and 44 spots displayed a significant sex by drought interaction effect. The results suggest that the different responses to drought stress detected between the sexes have a close relationship to the changes in the expression of sex‐dependent proteins, including, e.g. photosynthesis‐related proteins, homeostasis‐related proteins and stress response proteins. These proteins could contribute to a physiological advantage under drought, giving potential clues for understanding sexual differences in the performance of plants in different environments.     

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.     

Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana

High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro‐environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ‐on‐line 2‐D nano‐LC/MS) in a non‐model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high‐quality proteomic dataset consisted of 695 proteins. Using a cutoff of P < 0.05, 241 unique proteins with differential expression levels were identified between control and different inoculum sizes. Functional analysis showed that proteins participating in photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 10⁶ cells mL^?1, and lowest levels under 1 × 10⁷ cells mL^?1. Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up‐regulation under the illumination of 70–110 µmol m^?2 s^?1, compared to those of 40 µmol m^?2 s^?1. This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Biotechnol. Bioeng. 2013; 110: 773–784. © 2012 Wiley Periodicals, Inc.     

Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH₂PO₄ (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO₂ enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress.     

Rice lectin receptor-like kinase provides salinity tolerance by ion homeostasis

Sensing stress and activating the downstream signaling pathways is the imperative step for stress response. Lectin receptor-like kinase (LecRLK) is an important family that plays a key role in sensing stress conditions through lectin receptor and activates downstream signaling by kinase domain. We identified the role of OsLecRLK gene for salinity stress tolerance and hypothesized its role in Na⁺ extrusion from cell. OsLecRLK overexpression and downregulation (through artificial miRNA) transgenic lines were developed and its comparison with wild-type (WT) plants were performed overexpression transgenic lines showed better performance, whereas downregulation showed poor performance than WT. Lower accumulation of reactive oxygen species (ROS), malondialdehyde and toxic ion, and a higher level of proline, RWC, ROS scavengers in overexpression lines lead us to the above conclusion. Based on the relative expression of stress-responsive genes, ionic content and interactome protein, working model highlights the role of OsLecRLK in the extrusion of Na⁺ ion from the cell. This extrusion is facilitated by a higher expression of salt overly sensitive 1 (Na⁺/K⁺ channel) in overexpression transgenic line. Altered expression of stress-responsive genes and changed biochemical and physiological properties of cell suggests an extensive reprogramming of the stress-responsive metabolic pathways by OsLecRLK under stress condition, which could be responsible for the salt tolerance capability.     

Salinity-induced changes in protein expression in the halophytic plant Nitraria sphaerocarpa

Salinity is a major abiotic stress that inhibits plant growth and development. Plants have evolved complex adaptive mechanisms that respond to salinity stress. However, an understanding of how plants respond to salinity stress is far from being complete. In particular, how plants survive salinity stress via alterations to their intercellular metabolic networks and defense systems is largely unknown. To delineate the responses of Nitraria sphaerocarpa cell suspensions to salinity, changes in their protein expression patterns were characterized by a comparative proteomic approach. Cells that had been treated with 150 mM NaCl for 1, 3, 5, 7, or 9 days developed several stress-related phenotypes, including those affecting morphology and biochemical activities. Of ~1100 proteins detected in 2-DE gel patterns, 130 proteins showed differences in abundance with more than 1.5-fold when cells were stressed by salinity. All but one of these proteins was identified by MS and database searching. The 129 spots contained 111 different proteins, including those involved in signal transduction, cell rescue/defense, cytoskeleton and cell cycle, protein folding and assembly, which were the most significantly affected. Taken together, our results provide a foundation to understand the mechanism of salinity response.