Proteomic approach to study leaf proteins in a fast-growing tree species, Gmelina arborea Linn. Roxb

Foliar proteome studies have become highly significant for a comprehensive understanding of complex processes associated with plant growth and development. In the present study, we present a proteomic approach to analyze leaf proteins in an important timber-yielding and fast-growing forest tree species, Gmelina arborea Linn. Roxb. (Verbanaceae). Foliar protein analysis involved protein extraction, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF–TOF). From the 2-DE protein profile of Gmelina leaves, we identified and isolated 150 well-separated protein spots; among these, 64 protein spots were identified by mass spectrometric (MS/MS) analysis. These proteins were classified according to their involvement in basic biological functions, such as photosynthesis, amino acid metabolism, cytoskeleton, cell wall metabolism, stress-related proteins, redox maintenance, electron transport chain, phytohormone metabolism and protein translation and folding. Analytical variance was determined for the protein spots of samples from different plants. The present study is believed to provide a foundation for the use of leaf proteomics in addressing fundamental physiological and biochemical processes associated with growth and productivity of tree species such as Gmelina arborea.     

Comparative proteomic profiles of the soybean (Glycine max) root apex and differentiated root zone

The root apical meristem (RAM) is responsible for the growth of the plant root system. Because of the importance of root architecture in the performance of crop plants, we established a proteome reference map of the soybean root apex and compared this with the proteome of the differentiated root zone. The root apex samples contained the apical 1?mm of the root, comprising the RAM, quiescent center and root cap. We identified 342 protein spots from 550 excised proteins (～62%) of root apex samples by MALDI-TOF MS/MS analysis. All these proteins were also present in the differentiated root, but differed in abundance. Functional classification showed that the most numerous protein categories represented in the root were those of stress response, glycolysis, redox homeostasis and protein processing. Using DIGE, we identified 73 differentially accumulated proteins between root apex and differentiated root. Proteins overrepresented in the root apex belonged primarily to the pathways for protein synthesis and processing, cell redox homeostasis and flavonoid biosynthesis. Proteins underrepresented in the root apex were those of glycolysis, tricarboxylic acid metabolism and stress response. Our results highlight the importance of stress and defense response, redox control and flavonoid metabolism in the root apex.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases

By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50 mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20 mM) than for the wild (3–6 mM) cells.Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5–8 pH range. More differences in protein content (37–64 mg g^? 1 cell dry weight) and number of resolved spots (178–307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.     

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

拟南芥AtrbohD和AtrbohF缺失对叶蛋白质组的影响

拟南芥NADPH氧化酶AtrbohD和AtrbohF在脱落酸（abscisic acid,ABA）抑制主根伸长、ABA诱导气孔关闭以及植物应答干旱、盐及病菌侵染等逆境胁迫反应中发挥重要作用,但这2个蛋白亚基缺失对拟南芥（Arabidopsis thaliana）蛋白质组的影响还未见报道。我们以营养土中生长16 d的野生型及AtrbohD和AtrbohF双基因突变体atrbohD1/F1叶片为材料进行蛋白组学分析,在双向电泳图谱上可分辨出约1 000个蛋白点,且蛋白表达谱存在差异。选取42个显著差异蛋白点进行MALDI-TOF/TOF质谱鉴定,成功鉴定出20个差异蛋白,这些蛋白主要与氧化还原、能量代谢、蛋白代谢、转录和信号传导等相关,还有一些蛋白功能未知。     

Analysis of proteins in aerenchymatous seminal roots of wheat grown in hypoxic soils under waterlogged conditions

Hypoxia caused by waterlogging results in a severe loss of crop production. At the primary stage of wheat development, the seminal roots have strategies to survive under hypoxia through alternative metabolism coupling root anatomical modification. The present study used a model system of lysigenous aerenchymatous seminal roots from a representative seedling stage of wheat to elucidate the root physiology in response to soil hypoxia. Seminal roots characteristic with lysigenous aerenchyma tissues were developed in pot cultures for 7 days under two hypoxic conditions, water depths of 15 cm below and 3 cm above the soil surface. Proteins from the roots were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. The results showed that approximately 345 distinct protein spots were detected by 2-DE, 29 spots changed in the expression levels between the control and two hypoxic plants, and 10 spots exhibited a reproducible up- or down regulated fluctuation. The up-regulated proteins were thought to be involved in alteration in energy and redox status, defense responses and cell wall turnover. These results suggest the effects of soil hypoxia on the activity of the identified up-regulated proteins and their roles in alternative respiration and cell degeneration in wheat in order to gain metabolic adjustment under hypoxia stress.     

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m⁻¹ in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m⁻¹ solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H⁺-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H⁺-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.     

Comparative Proteomic Analysis Reveals the Cross-Talk between the Responses Induced by H2O2 and by Long-Term Rice Black-Streaked Dwarf Virus Infection in Rice

Hydrogen peroxide (H₂O₂) could be produced during the plant-virus compatible interaction. However, the cell responses regulated by the enhanced H₂O₂ in virus infected plant are largely unknown. To make clear the influence of Rice black-streaked dwarf virus (RBSDV) infection on H₂O₂ accumulation, we measured the content of H₂O₂ and found the H₂O₂ level was increased in rice seedlings inoculated with RBSDV. To reveal the responses initiated by the enhanced H₂O₂ during plant-virus interaction, the present study investigated the global proteome changes of rice under long-term RBSDV infection. Approximately 1800 protein spots were detected on two-dimensional electrophoresis (2-DE) gels. Among them, 72 spots were found differently expressed, of which 69 spots were successfully identified by MALDI-TOF/TOF-MS. Furthermore, the differentially expressed proteins induced by RBSDV infection were compared to that induced by H₂O₂. 19 proteins corresponding to 37 spots, which were differentially expressed under RBSDV infection, were observed differentially expressed under H₂O₂ stress as well. These overlapping responsive proteins are mainly related to photosynthesis, redox homeostasis, metabolism, energy pathway, and cell wall modification. The increased H₂O₂ in RBSDV infected plant may produce an oxidative stress, impair photosynthesis, disturb the metabolism, and eventually result in abnormal growth. The data provide a new understanding of the pivotal role of H₂O₂ in rice-RBSDV compatible interaction.     

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H₂O₂ level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.     

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light‐ and dark‐grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress‐related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light‐grown cells were more sensitive to heavy metal toxicity than dark‐grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism‐related proteins increased in relative abundance both in light‐ and dark‐grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti‐oxidative processes were decreased both in light‐ and dark‐grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light‐dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.     

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.     

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione‐S‐transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.