拟南芥神经酰胺酶基因对氧化胁迫的响应

以拟南芥哥伦比亚生态型（Col）和神经酰胺酶突变体为实验材料,通过对突变体的一系列生理生化指标的测定,来研究拟南芥神经酰胺酶基因（AtCER）对H2O2的响应。利用PCR和Northern blot获得了9个AtCER纯合单突变体。不同浓度H2O2处理野生型和突变体后,发现突变体对H2O2的反应比野生型更加敏感。H2O2处理后突变体叶片出现比野生型更严重的黄化现象和坏死斑点,总叶绿素含量也比野生型下降的更快,电导率测定也发现突变体比野生型的电导率增加得更多。抗氧化酶活性的分析结果发现H2O2处理后,突变体的抗氧化酶活性比野生型提高了1．5～3倍。上述研究结果说明AtCER参与了H2O2诱导的氧化胁迫反应。     

拟南芥GLP13基因在植物抗氧化胁迫响应中的作用

类萌发素蛋白（germin-like protein, GLPs）是一类与小麦萌发素序列相似性较高、位于胞外基质的可溶性糖蛋白, 在植物的生长发育阶段以及对生物和非生物胁迫的应答中起着重要的作用。为了研究GLP13基因的生理功能, 我们分离并鉴定了GLP13的敲减突变体glp13, 同时构建了其超表达植株35S：：GLP13。用甲基紫精（methyl viologen, MV）处理2种不同基因型和野生型（WT）植株, 结果发现, 与野生型相比, 突变体glp13子叶变绿率较低, 主根生长受抑制较明显; 而超表达植株35S：：GLP13子叶变绿率较高, 主根生长的受抑制程度较WT轻。用MV处理2周的35S：：GLP13植株, 其叶绿素荧光参数Fv/Fm 的下降较野生型对照缓慢。半定量RT-PCR分析结果表明, 与野生型相比, 经MV处理4小时后的35S：：GLP13中抗氧化酶系基因FSD1的表达上调, 而CAT1、CSD1和UGT71C1的表达水平在35S：：GLP13、glp13和野生型植株三者之间没有明显差异。以上结果表明GLP13基因在拟南芥抗氧化胁迫响应中起重要作用。     

NaCI胁迫对盐芥和拟南芥光合作用的影响

本研究检测了与盐芥(Ghellungiella halophila)和拟南芥(Arabidopsis thaliana)光合作用相关的叶绿素、净光合速率(photosynthetic rate,Pn)、气孔导度(stomatal conductance,Gs)、胞间隙CO2浓度以及叶绿素荧光参数等指标,观察到随着NaCl浓度逐渐增加,盐芥的叶绿素a／b值(Chl a／Chl b)、类胡萝卜素／总叶绿素值(Car／Chl)显著高于拟南芥,且二比值变化幅度较小并保持较高水平。盐胁迫下拟南芥净光合速率下降、气孔导度下降和胞间CO2浓度减小。气孔因素是引起拟南芥光合能力下降的主要因素。叶绿素荧光参数的变化表明,50-200 mmol·L-1NaCl降低拟南芥叶绿体对光能的吸收能力,而且降低叶绿体的光化学活性,使电子传递速率和光能转化效率大幅度下降,造成光能转化为化学能的过程受阻,进一步加剧了光合放氧和碳同化能力的降低。而50-200 mmol·L-1NaCl胁迫没有使盐芥的光合作用受到不良影响。     

NaCl 胁迫对盐芥和拟南芥光合作用的影响

本研究检测了与盐芥(Ghellungiella halophila)和拟南芥(Arabidopsis thaliana)光合作用相关的叶绿素、净光合速率(photosynthetic rate, Pn)、气孔导度(stomatal conductance, Gs)、胞间隙CO2浓度以及叶绿素荧光参数等指标, 观察到随着NaCl浓度逐渐增加, 盐芥的叶绿素a/b值(Chl a/Chl b)、类胡萝卜素/总叶绿素值(Car/Chl)显著高于拟南芥, 且二比值变化幅度较小并保持较高水平。盐胁迫下拟南芥净光合速率下降、气孔导度下降和胞间CO2浓度减小。气孔因素是引起拟南芥光合能力下降的主要因素。叶绿素荧光参数的变化表明, 50-200 mmol.L-1 NaCl降低拟南芥叶绿体对光能的吸收能力, 而且降低叶绿体的光化学活性, 使电子传递速率和光能转化效率大幅度下降,造成光能转化为化学能的过程受阻,进一步加剧了光合放氧和碳同化能力的降低。而50-200 mmol.L-1 NaCl 胁迫没有使盐芥的光合作用受到不良影响。     

Intracellular Solutes,Photosynthesis and Respiration of the Green Alga Blidingia minima in Response to Salinity Stress

The effects of different salinities ranging from 7–68‰ on the internal inorganic and organic solute concentrations, and on the photosynthesis and respiration have been investigated in the green alga Bladingia minima (Näg. ex Kütz.) Kylin. The levels of the main osmotic solutes K⁺, sucrose and proline increased with increasing salinities and vice versa, while Na⁺, Mg²⁺, Cl^? and PO^3–₄ played a minor role in the osmotic acclimation. In contrast to related Enteromorpha species, B. minima exhibited high NO^?₃ concentrations, which decreased under hypo- and hypersaline conditions. B. minima differs also from Enteromorpha by accumulating the tertiary sulphonium compound DMSP in osmotically significant amounts under gentle hypersaline conditions. B. minima revealed typical characteristics of a “sun-plant” having a high light compensation point together with a saturation of photosynthesis at high photon flux densities. The alga showed a broad photosynthetic stability under osmotic stress; only with extreme hypersaline conditions was photosynthesis partly inhibited. The rate of respiration remained constant in hypersaline media, and was stimulated under hyposaline conditions.     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

植物铁蛋白与氧化胁迫应激

植物铁蛋白是植物体重要的铁调节蛋白。许多研究表明植物铁蛋白与氧化胁迫抗性之间具有较强关联。植物铁蛋白不仅能抵御高铁产生的氧化毒性,在很多氧化胁迫及环境胁迫抗性中也发挥作用。对植物铁蛋白在氧化及逆境胁迫中的应激加以综述,为铁蛋白在生物工程领域的应用提供理论依据。     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1–3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4–8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and label-free quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS)¹ has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9–12). On the other hand, label-free quantitation has gained momentum in recent years (13–15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quantitation. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides. Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples (20). One solution to large-scale quantitation without any isotope labeling is pseudo internal standard approach (21), which selects endogenous house-keeping proteins as the internal standard so that no spike-in reagent is required. However, finding a good pseudo internal standard remains a challenge for phosphoproteome samples. Spike-in experiments are an alternative way to improve normalization profile. Some internal standard peptides such as MassPREP^TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22–24). There are two major normalization procedures. In one approach, sample peptides were normalized to the total peak intensity of spike-in peptides (25). Alternatively, the digested peptides were compared at first and the normalization factor was determined in different ways such as the median (26) or average of ratios (27). However, spiking an identical amount of standard proteins into phosphoproteomic samples before protein digestion may not be compatible with phosphoproteomic analyses which typically require a phosphopeptide enrichment step. Spectral counting has been extensively applied in large sets of proteomic samples because of its simplicity but the method is often not reliable for the quantitation of phosphoproteins, which are typically identified by single phosphopeptides with few spectra (28–30). Many software packages have been implemented to support the wide variety of those quantitation techniques, including commercial platforms such as Progenesis LC-MS^TM, Mascot Distiller^TM, PEAKS Q^TM, etc., as well as open-source software packages including MaxQuant (31), PEPPeR (32), Skyline (33), etc.In this study, we have devised a novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency (Fig. 1). The approach employs synthetic peptide libraries as the internal standard. These peptides were prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides were designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity is performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (34). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide.Open in a separate windowFig. 1.Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R²) and accuracy (%Acc).Water deficit and salinity causes osmotic stress, which is a major environmental factor limiting plant agricultural productivity. Osmotic stress rapidly changes the metabolism, gene expression and development of plant cells by activating several signaling pathways. Several protein kinases have been characterized as key components in osmotic stress signaling. Arabidopsis histidine kinase AHK1 can complement the histidine kinase mutant yeast, which can act as the osmosensor in yeast (35). Overexpression of AHK1 gene increases the drought tolerance of transgenic plants in Arabidopsis (36). Similar to yeast, the MAPK kinase cascade is also involved in osmotic stress response in plants. It is reported that AtMPK3, AtMPK6, and tobacco SIPK can be activated by NaCl or mannitol, and play positive roles in osmotic signaling (37, 38). MKK7 and MKKK20 may act as the up-stream kinase in the kinase cascade (39). Involvement of some calcium-dependent protein kinases, such as AtCPK21, AtCPK6, and OsCPK7 (CDPK) in osmotic stress signaling has also been reported (40–42). Another kinase family, SNF1-related protein kinase (SnRK) 2, also participates in osmotic stress response. In Arabidopsis, there are ten members in the SnRK2 family. Five from the ten SnRK2s, SnRK2, 3, 6, 7, and 8, can be activated by abscisic acid (ABA) and play central roles in ABA-receptor coupled signaling (43, 44). Furthermore, all SnRK2s except SnRK2.9 can be activated by NaCl or mannitol treatment (43). The decuple mutant of SnRK2 showed a strong osmotic hypersensitive phenotype (45). It is proposed that protein kinases including MAPK and SnRK2s have a critical function in osmotic stress (46), but the detailed mechanism and downstream substrates or target signal components are not yet clarified. We applied, therefore, the LAXIC approach with a self-validating method (47) to profile the osmotic stress-dependent phosphoproteome in Arabidopsis by quantifying phosphorylation events before and after mannitol treatment. Among a total of over 2000 phosphopeptides, more than 400 of them are dependent on osmotic stress. Those phosphoproteins are present on enzymes participating in signaling networks that are involved in many processes such as signal transduction, cytoskeleton development, and apoptosis. Overall, LAXIC represents a powerful tool for label-free quantitative phosphoproteomics.     

Response of Melanthera biflora to Salinity and Water Stress

Melanthera biflora (Asteraceae) is a moderately salt-tolerantplant from the Indo-Pacific region. In laboratory studies itsgrowth was inhibited by salt above 50 mol m^–3, but itwas able to survive salinities approaching that of seawater,namely 400 mol m^–3. Shoot potassium concentrations weremaintained over a range of salinities up to 400 mol m^–3,while sodium and chloride accumulation followed closely theincrease in external osmotic pressure. In contrast, the increasein osmotic pressure of the leaf sap of Melanthera biflora, subjectedto water stress, was due mainly to a decrease in the ratio offresh weight/dry weight. 3-dimethylsulphoniopropionate (3-DMSP)and glycinebetaine were identified by fast atom bombardmentmass and ¹H -NMR spectroscopy, with 3-DMSP being the main oniumcompound and glycinebetaine absent in some accessions. Onium(quaternary ammonium and/or tertiary sulphonium) compounds andproline increased during salt and water stress due mainly toa decrease in the fresh weight/dry weight ratio of tissue, althoughpart of the increase in salt-stressed tissue was due to an increasein the accumulation of the onium compound. This salt-inducedincrease in 3-DMSP was inhibited in conditions of low sulphursupply and there was no compensatory increase in proline. Key words: Melanthera biflora, Asteraceae, salinity, glycinebetaine, 3-dimethylsulphonioproprionate     

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.     

Changes in the Metabolome of Two Soybean Genotypes under Drought Stress

Russian Journal of Plant Physiology - Soybean is the world’s leading economic oilseed crop. Drought stress is a major constraint on the growth and yield stability of soybean. Here, wild...     

氧化应激下植物线粒体自噬分析

线粒体自噬,是指通过选择性的识别并清除损伤、衰老及功能紊乱的线粒体,对维持细胞内线粒体质量和数量的平衡产生了重要作用。与动物和酵母中线粒体自噬的研究进展相比,植物线粒体自噬的途径及具体调控机制尚不明确。基于GFP标签,本文探究了氧化胁迫下植物线粒体自噬发生情况。研究发现甲基紫精诱导线粒体在液泡中积累,并呈现两种状态:1) GFP小体包含的线粒体; 2)不含GFP的线粒体。本研究发展的GFP标签策略可为植物线粒体自噬关键调控因子的筛选提供借鉴。     

Characterization of the Nrt2.6 Gene in Arabidopsis thaliana: A Link with Plant Response to Biotic and Abiotic Stress

The high affinity nitrate transport system in Arabidopsis thaliana involves one gene and potentially seven genes from the NRT1 and NRT2 family, respectively. Among them, NRT2.1, NRT2.2, NRT2.4 and NRT2.7 proteins have been shown to transport nitrate and are localized on the plasmalemma or the tonoplast membranes. NRT2.1, NRT2.2 and NRT2.4 play a role in nitrate uptake from soil solution by root cells while NRT2.7 is responsible for nitrate loading in the seed vacuole. We have undertaken the functional characterization of a third member of the family, the NRT2.6 gene. NRT2.6 was weakly expressed in most plant organs and its expression was higher in vegetative organs than in reproductive organs. Contrary to other NRT2 members, NRT2.6 expression was not induced by limiting but rather by high nitrogen levels, and no nitrate-related phenotype was found in the nrt2.6-1 mutant. Consistently, the over-expression of the gene failed to complement the nitrate uptake defect of an nrt2.1-nrt2.2 double mutant. The NRT2.6 expression is induced after inoculation of Arabidopsis thaliana by the phytopathogenic bacterium Erwinia amylovora. Interestingly, plants with a decreased NRT2.6 expression showed a lower tolerance to pathogen attack. A correlation was found between NRT2.6 expression and ROS species accumulation in response to infection by E. amylovora and treatment with the redox-active herbicide methyl viologen, suggesting a probable link between NRT2.6 activity and the production of ROS in response to biotic and abiotic stress.