Proteomic analysis of salt stress-responsive proteins in rice root

Salt stress is one of the major abiotic stresses in agriculture worldwide. We report here a systematic proteomic approach to investigate the salt stress-responsive proteins in rice (Oryza sativa L. cv. Nipponbare). Three-week-old seedlings were treated with 150 mM NaCl for 24, 48 and 72 h. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis. More than 1100 protein spots were reproducibly detected, including 34 that were up-regulated and 20 down-regulated. Mass spectrometry analysis and database searching helped us to identify 12 spots representing 10 different proteins. Three spots were identified as the same protein, enolase. While four of them were previously confirmed as salt stress-responsive proteins, six are novel ones, i.e. UDP-glucose pyrophosphorylase, cytochrome c oxidase subunit 6b-1, glutamine synthetase root isozyme, putative nascent polypeptide associated complex alpha chain, putative splicing factor-like protein and putative actin-binding protein. These proteins are involved in regulation of carbohydrate, nitrogen and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. This study gives new insights into salt stress response in rice roots and demonstrates the power of the proteomic approach in plant biology studies.     

Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold 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.     

Proteomic Analysis of Salt-Responsive Proteins in the Leaves of Mangrove Kandelia candel during Short-Term Stress

Salt stress is a major abiotic stress that limits crop productivity in many regions of the world. A comparative proteomic approach to identify salt stress-responsive proteins and to understand the molecular mechanisms was carried out in the woody halophyte Kandelia candel. Four-leaf-old K. candel seedlings were exposed to 150 (control), 300, 450, and 600 mM NaCl for 3 days. Proteins extracted from the leaves of K. candel seedlings were separated by two-dimensional gel electrophoresis (2-DE). More than 900 protein spots were detected on each gel, and 53 differentially expressed protein spots were located with at least two-fold differences in abundance on 2-DE maps, of which 48 were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). The results showed that K. candel could withstand up to 450 mM NaCl stress by up-regulating proteins that are mainly involved in photosynthesis, respiration and energy metabolism, Na⁺ compartmentalization, protein folding and assembly, and signal transduction. Physiological data, including superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities, hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂ ⁻) contents, as well as Na⁺ content and K⁺/Na⁺ ratios all correlated well with our proteomic results. This study provides new global insights into woody halophyte salt stress responses. Identification of differentially expressed proteins promotes better understanding of the molecular basis for salt stress reduction in K. candel.     

Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress

Salt stress is one of the major abiotic stresses in agriculture worldwide, especially in the Mediterranean area. We report here a proteomic approach to investigate the salt stress-responsive proteins in grapevine (Vitis vinifera). Two-dimensional electrophoresis (2-DE) was used to analyze the proteome of the salt-tolerant Tunisian grapevine cultivar Razegui, subjected to a supply of 100mm NaCl over 15d. Analysis of 2-DE gels derived from stressed plants revealed more than 800 reproducibly detected protein spots, with 48 proteins displaying a differential expression pattern, including 32 up-regulated, 9 down-regulated and 7 new protein spots induced after salt treatment. The presence of stress-responsive proteins in the different plant organs suggests that salt spreads systemically. Edman degradation analysis and database searching aided us in identifying a major protein GP. Database analysis revealed that this peptide has a 98% sequence similarity with a pathogenesis-related (PR) protein 10 (V. vinifera). A full-length cDNA encoding the GP protein was isolated from grapevine salt-stressed leaves and sequenced. The predicted protein contained 158 amino acids and showed 98% identity with PR10 protein of of V. vinifera (accession no. Cac16165).     

Identification of differentially expressed salt-responsive proteins in roots of two perennial grass species contrasting in salinity tolerance

This study was designed to identify physiological responses and differential proteomic responses to salinity stress in roots of a salt-tolerant grass species, seashore paspalum (Paspalum vaginatum), and a salt-sensitive grass species, centipedegrass (Eremochloa ophiuroides). Plants of both species were exposed to salinity stress by watering the soil with 300 mM NaCl solution for 20 d in a growth chamber. The 2-DE analysis revealed that the abundance of 8 protein spots significantly increased and 14 significantly decreased in seashore paspalum, while 19 and 16 protein spots exhibited increase and decrease in abundance in centipedegrass, respectively. Eight protein spots that exhibited enhanced abundance in seashore paspalum under salinity stress were subjected to mass spectrometry analysis. Seven protein spots were successfully identified, they are peroxidase (POD, 2.36-fold), cytoplasmic malate dehydrogenase (cMDH, 5.84-fold), asorbate peroxidase (APX, 4.03-fold), two mitochondrial ATPSδ chain (2.26-fold and 4.78-fold), hypothetical protein LOC100274119 (5.01-fold) and flavoprotein wrbA (2.20-fold), respectively. Immunblotting analysis indicated that POD and ATPSδ chain were significantly up-regulated in seashore paspalum at 20 d of salinity treatment while almost no expression in both control and salt treatment of centipedegrass. These results indicated that the superior salinity tolerance in seashore paspalum, compared to centipedegrass, could be associated with a high abundance of proteins involved in ROS detoxification and energy metabolism.     

Proteome Dynamics and Physiological Responses to Short-Term Salt Stress in Brassica napus Leaves

Salt stress limits plant growth and crop productivity and is an increasing threat to agriculture worldwide. In this study, proteomic and physiological responses of Brassica napus leaves under salt stress were investigated. Seedlings under salt treatment showed growth inhibition and photosynthesis reduction. A comparative proteomic analysis of seedling leaves exposed to 200 mM NaCl for 24 h, 48 h and 72 h was conducted. Forty-four protein spots were differentially accumulated upon NaCl treatment and 42 of them were identified, including several novel salt-responsive proteins. To determine the functional roles of these proteins in salt adaptation, their dynamic changes in abundance were analyzed. The results suggested that the up-accumulated proteins, which were associated with protein metabolism, damage repair and defense response, might contribute to the alleviation of the deleterious effect of salt stress on chlorophyll biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves. This study will lead to a better understanding of the molecular basis of salt stress adaptation in Brassica napus and provides a basis for genetic engineering of plants with improved salt tolerance in the future.     

Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes

Salinity is one of the major environmental constraints limiting yield of crop plants in many semi-arid and arid regions around the world. To understand responses in soybean seedling to salt stress at proteomic level, the extracted proteins from seedling leaves of salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68 under 150 mM NaCl stress for 1, 12, 72 and 144 h, respectively, were analyzed by 2-DE. Approximately 800 protein spots were detected on 2-DE gels. Among them, 91 were found to be differently expressed, with 78 being successfully identified by MALDI-TOF-TOF. The identified proteins were involved in 14 metabolic pathways and cellular processes. Based on most of the 78 salt-responsive proteins, a salt stress-responsive protein network was proposed. This network consisted of several functional components, including balancing between ROS production and scavenging, accelerated proteolysis and reduced biosynthesis of proteins, impaired photosynthesis, abundant energy supply and enhanced biosynthesis of ethylene. Salt-tolerant genotype Lee 68 possessed the ability of higher ROS scavenging, more abundant energy supply and ethylene production, and stronger photosynthesis than salt-sensitive genotype Jackson under salt stress, which may be the major reasons why it is more salt-tolerant than Jackson.     

Proteomic analysis of salinity-stressed <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revealed differential suppression and induction of a large number of important housekeeping proteins

Salinity stress is one of the most common abiotic stresses that hamper plant productivity worldwide. Successful plant adaptations to salt stress require substantial changes in cellular protein expression. In this work, we present a 2-DE-based proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, subjected to 300 mM NaCl for 2 h. Results showed that, in addition to the protein spots that showed partial up- or down-regulation patterns, a number of proteins were exclusively present in the proteome of the control cells, but were absent from the salinity-stressed samples. Conversely, a large number of proteins exclusively appeared in the proteome of the salinity-stressed samples. Of those exclusive proteins, we could successfully identify, via LC–MS/MS, 18 spots uniquely present in the control cells and 99 spots specific to NaCl-treated cells. Interestingly, among the salt-exclusive protein spots, we identified several important housekeeping proteins like molecular chaperones and proteins of the translation machinery, suggesting that they may originate from post-translational modifications rather than from de novo biosynthesis. The possible role and the salt-specific modification of these proteins by salinity stress are discussed.     

Proteomic and phosphoproteomic analyses of NaCl stress-responsive proteins in Arabidopsis roots

Salt is one of the major abiotic stresses limiting the productivity and the geographical distribution of crops. To gain a better understanding of NaCl stress responses in model plant Arabidopsis roots, the protein changes in the abundance (Coomassie Brilliant Blue R-350 stain) and phosphorylation (Pro-Q Diamond stain) were examined using two-dimensional electrophoresis coupled with mass spectrometry (MS). Seventeen unique proteins differentially changed in abundance, phosphorylation, or both in response to NaCl. Nonsynchronous differences were found between total proteins and phosphorylated proteins. Protein synthesis, proteolysis, post-translational modifications, and isoforms might cause the differential protein redundancies. The identified proteins are involved in binding, catalysis, signal transduction, transport, metabolisms of cell wall and energy, and reactive oxygen species (ROS) scavenging and defense. These protein changes provide new avenues of investigation into the underlying salt stress response in Arabidopsis roots and demonstrate the advantages of proteomic approach in plant biology studies.     

Comparative proteomic analysis provides new insights into chilling stress responses in rice

Low temperature is one of the major abiotic stresses limiting the productivity and the geographical distribution of many important crops. To gain a better understanding of chilling stress responses in rice (Oryza sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis. Three-week-old rice seedlings were treated at 6 degrees C for 6 or 24 h and then recovered for 24 h. Chilling treatment resulted in stress phenotypes of rolling leaves, increased relative electrolyte leakage, and decreased net photosynthetic rate. The temporal changes of total proteins in rice leaves were examined using two-dimensional electrophoresis. Among approximately 1,000 protein spots reproducibly detected on each gel, 31 protein spots were down-regulated, and 65 were up-regulated at least at one time point. Mass spectrometry analysis allowed the identification of 85 differentially expressed proteins, including well known and novel cold-responsive proteins. Several proteins showed enhanced degradation during chilling stress, especially the photosynthetic proteins such as Rubisco large subunit of which 19 fragments were detected. The identified proteins are involved in several processes, i.e. signal transduction, RNA processing, translation, protein processing, redox homeostasis, photosynthesis, photorespiration, and metabolisms of carbon, nitrogen, sulfur, and energy. Gene expression analysis of 44 different proteins by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. In conclusion, our study provides new insights into chilling stress responses in rice and demonstrates the advantages of proteomic analysis.     

Comparative proteomic analysis of alfalfa revealed new salt and drought stress-related factors involved in seed germination

Salinity and drought are two major environmental factors that limit the growth and yield of many forage crops in semi-arid and arid regions. Alfalfa (Medicago sativa L.) is one of the most important forage crops in many countries. We aim to investigate the molecular mechanisms of alfalfa in response to salt and drought stresses in this study. Physiological and proteomic analyses were applied to examine the Zhongmu NO.3 alfalfa seed germination stage with 200 mM NaCl and 180 g·L^?1 polyethylene glycol (PEG) treatments. The germination ability of the seed and the accumulation of osmotic solutes were quite different between the NaCl and PEG treatments. More than 800 protein spots were detected by proteomics technology on two-dimensional electrophoresis (2-DE) gels. The abundance of twenty-eight proteins were decreased or increased after salt and drought stress. Seventeen of these proteins were identified and classified into six functional categories through mass spectrometry (MS). The six groups involved in salt- and PEG-mediated stress included defense response, energy metabolism, protein synthesis and degradation, oxidative stress, carbohydrate metabolism-associated proteins, and unknown proteins. We discovered that some proteins related to carbohydrate metabolism and energy production increased in abundance under salt- and PEG-mediated drought stress. This demonstrates a common mechanism of energy consumption during abiotic stresses. Further study of these proteins with unknown function will provide insights into the molecular mechanisms of abiotic stress and the discovery of new candidate markers.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

Proteome analysis of soybean roots subjected to short-term drought stress

Drought is one of the most important constraints on the growth and productivity of many crops, including soybeans. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we carried out a proteome analysis in combination with physiological analyses of soybean roots subjected to severe but recoverable drought stress at the seedling stage. Drought stress resulted in the increased accumulation of reactive oxygen species and subsequent lipid peroxidation. The proline content increased in drought-stressed plants and then decreased during the period of recovery. The high-resolution proteome map demonstrated significant variations in about 45 protein spots detected on Comassie briliant blue-stained 2-DE gels. Of these, 28 proteins were identified by mass spectrometry; the levels of 5 protein spots were increased, 21 were decreased and 2 spots were newly detected under drought condition. When the stress was terminated by watering the plants for 4 days, in most cases, the protein levels tended towards the control level. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death, and they contribute to the molecular mechanism of drought tolerance in soybean plants. Analysis of protein expression patterns revealed that proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles for soybean plant drought adaptation. The identification of these proteins provides new insight that may lead to a better understanding of the molecular basis of the drought stress responses.     

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques.