Proteome analysis of sugar beet (Beta vulgaris L.) elucidates constitutive adaptation during the first phase of salt stress

Salinity is one of the major stress factors responsible for growth reduction of most of the higher plants. In this study, the effect of salt stress on protein pattern in shoots and roots of sugar beet (Beta vulgaris L.) was examined. Sugar beet plants were grown in hydroponics under control and 125 mM salt treatments. A significant growth reduction of shoots and roots was observed. The changes in protein expression, caused by salinity, were monitored using two-dimensional gel-electrophoresis. Most of the detected proteins in sugar beet showed stability under salt stress. The statistical analysis of detected proteins showed that the expression of only six proteins from shoots and three proteins from roots were significantly altered. At this stage, the significantly changed protein expressions we detected could not be attributed to sugar beet adaptation under salt stress. However, unchanged membrane bound proteins under salt stress did reveal the constitutive adaptation of sugar beet to salt stress at the plasma membrane level.     

Differential Two-Dimensional Protein Patterns as Related to Tissue Specificity and Water Conditions in Brassica napus var oleifera Root System

Differential two-dimensional protein patterns as related to tissue specificity and water conditions were investigated within Brassica napus var oleifera root system. The different parts of the root system (tap root, lateral roots, and drought-induced short roots) were analyzed under various moisture regimes (regular watering at field capacity, progressive drought stress, and rewatering). Tissue specificity was evident from 25 differences in protein patterns (qualitative and quantitative) between well-watered lateral and tap roots. Twice as many polypeptides (52) were drought-affected and the response to the water stress was shown to be similar in both root types. In addition, more than half of the polypeptides detected as organ-specific were affected by drought. Based upon the trend of variation observed under drought and rehydration, three categories of polypeptides could be defined that might be differently involved in drought susceptibility or tolerance. A highly differentiated protein pattern characterized the drought-induced short roots. This pattern appeared as far from the watered as from the water-stressed normal roots. In particular, 13 unique polypeptides were detected which could be relevant to their adaptive morphogenesis and/or their specific drought tolerance induction. Upon rehydration, their polypeptide pattern and their specific morphology returned to a normal well-watered lateral root type.     

Salt Stress Proteins Induced in Listeria monocytogenes

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).     

Comparative protein profiles of <Emphasis Type="Italic">Butea superba</Emphasis> tubers under seasonal changes

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Proteomic Changes in the Stem of Wild <Emphasis Type="Italic">Pteridium aquilinum</Emphasis> During Development

Lignification is one of the most crucial factors affecting the edible value of the stem of wild Pteridium aquilinum. To investigate the probable mechanism of lignification, the changes in protein profiles in the stem of wild P. aquilinum during its development were investigated by means of two-dimensional electrophoresis technology. The two-dimensional electrophoresis results revealed that there were twenty-seven differential proteins, twenty-four proteins of which were identified by MALDI-TOF/TOF. We classified these twenty-four proteins into six functional categories: photosynthesis (8, 33.3 %); respiratory metabolism (4, 16.7 %); stress response and defence (6, 25.0 %); cell structure (1, 4.2 %); phenylpropanoid metabolism (4, 16.6 %) and unclassified protein (1, 4.2 %). According to the functional analysis of these differentially expressed proteins, we concluded that photosynthesis was enhanced during P. aquilinum’s development and sugars generated from photosynthesis were partially metabolized through the glycolysis pathway and phosphopentose pathway, respectively, thus producing the precursors for lignin biosynthesis. The up-regulation of caffeoyl-CoA-O-methyl-transferase and SAM synthetase in abundance and the down-regulation of chalcone synthase can be directly responsible for lignification during stem development. This experiment is useful for understanding the biochemical mechanisms of the lignification process of P. aquilinum during its development.     

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS–PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS–PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.     

Cold-stress response in the amphibian oocyte: changes in synthesis and nucleocytoplasmic distribution of some proteins

In vivo cold stress was found to induce characteristic changes in the protein synthesis pattern of Pleurodeles waltl oocytes, as analyzed by two-dimensional gel electrophoresis. The nature of the response varied with the duration and intensity of stress. After a short period of cold stress (12 h at 8°C), synthesis and intracellular distribution of polypeptides were dramatically disturbed. There occured: 1) a reduction in synthesis of several polypeptides, including two major polypeptides (54-kDa and 47-kDa); 2) changes in distribution of polypeptides in oocyte, ie some polypeptides (185- and 96-kDa) were blocked in the cytoplasm, while other polypeptides (82-, 74-, 72- and 68-kDa, actin and nucleoplasmin) continued to enter the nucleus, but were quantitatively reduced; 3) no changes in the distribution of two nuclear polypeptides (53- and 43-kDa); 4) changes in the relative quantities of β- and γ-actin and preferential migration of γ-actin towards the nucleus. After a long period of in vivo cold stress (5 days at 8°C), we noted a partial recuperation of synthesis and nuclear migration (except for a 96-kDa polypeptide), but a persistent perturbation at the level of actin. For more drastic stress conditions (4°C), such a recuperation of protein synthesis was never observed.     

Analysis of Lupinus albus Leaf Apoplastic Proteins in Response to Boron Deficiency

Boron is essential for plant development and although its precise functions are not fully understood there is evidence for its importance in the extracellular matrix. So we have analysed by two-dimensional gel electrophoresis the effect of B deficiency in the soluble apoplast proteins of Lupinus albus leaf. Twenty-three polypeptide spots varied significantly between the control and the B deficiency patterns. Of these polypeptides only 9 could be identified by mass spectrometry techniques: PR-1 like protein, β-1,3-glucanases, class III chitinases, thaumatin like proteins and an expansin-like protein, all of them being involved in plant defence mechanisms. Only PR-1 like protein was de novo expressed under B deficiency, while the remaining proteins also responded to water stress. Although general response mechanisms seem to be triggered by both B and water stress, the pattern of protein expression was distinct, suggesting that under B deficiency specific regulatory mechanisms may be induced.     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

Rearrangement of cortex proteins constitutes an osmoprotective mechanism in Dictyostelium.

Dictyostelium responds to hyperosmotic stress of 400 mOsm by a rapid reduction of its cell volume to 50%. The reduced cell volume is maintained as long as these osmotic conditions prevail. Dictyostelium does not accumulate compatible osmolytes to counteract the osmotic pressure applied. Using two-dimensional gel electrophoresis, we demonstrate that during the osmotic shock the protein pattern remains unaltered in whole-cell extracts. However, when cells were fractionated into membrane and cytoskeletal fractions, alterations of specific proteins could be demonstrated. In the crude membrane fraction, a 3-fold increase in the amount of protein was measured upon hyperosmotic stress. In the cytoskeletal fraction, the proteins DdLIM and the regulatory myosin light chain (RMLC) were shown to be regulated in the osmotic stress response. The elongation factors eEF1alpha (ABP50) and eEF1beta were found to increase in the cytoskeletal fraction, suggesting a translational arrest upon hyperosmotic stress. Furthermore, the two main components of the cytoskeleton, actin and myosin II, are phosphorylated as a consequence of the osmotic shock, with a tyrosine residue as the phosphorylation site on actin and three threonines in the case of the myosin II heavy chain.     

A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of l-lactic acid

The growth rate and maximum biomass of Bacillus coagulans 2–6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2–6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress.     

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Identification of dimedone-trapped sulfenylated proteins in plants under stress

In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H₂O₂) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis. We conclude that dimedone can penetrate the cell wall, does not stress plants, and can “read” the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H₂O₂ was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H₂O₂-stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants.     

Stress- and Growth Phase-Associated Proteins of Clostridium acetobutylicum

The response of Clostridium acetobutylicum ATCC 4259 to the stresses produced by a temperature upshift from 28°C to 45°C and by exposure of the organisms to 0.1% n-butanol or to air was examined by analysis of pulse-labeled proteins. The stress response was the induction of the synthesis of a number of proteins, some of which were elicited by the three forms of stress. Eleven heat shock proteins were identified by two-dimensional electrophoresis, as were two proteins whose synthesis was heat sensitive. In the absence of applied stress, the synthesis of four proteins was found to be associated with the growth phase in batch culture; three of these proteins had a higher rate of de novo synthesis when the cells entered the solvent production phase. One of the stress-induced proteins, hsp74, was partially purified an found to be immunologically related to Escherichia coli heat shock protein Dnak. The similarities of the proteins induced at the onset of solventogenesis and by stress suggest a relationship between the two processes.     

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.     

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.     

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.     

Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.Keyword: Abiotic stress, Cinnamoyl CoA reductase, Developing seedlings, Leucaena leucocephala     

A Possible Proteome-level Explanation for Differences in Virulence of Two Isolates of a Fungal Pathogen Alternaria brassicae

Brassica napus (canola) is an important oilseed crop in many countries throughout the world including Canada. One of the major constraints on canola productivity is blackspot disease caused by the necrotrophic phytopathogenic fungus Alternaria brassicae. Two isolates of A. brassicae with significant differences in virulence have been characterized at the proteomelevel. The morphological observations indicated the Ontario isolate to be more virulent by virtue of increased disease severity score as compared to the UAMH7476 isolate. This was further confirmed through histological observations that indicated extensive colonization of the host tissue by the highly virulent isolate. Mycelial protein profiles of two differentially virulent A. brassicae isolates were compared using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) in order to identify proteins that may be responsible for the observed differences. The investigation suggested several differences in the mycelial proteomes of the two isolates. The proteins that were significantly abundant in the more virulent isolate included a protein with conserved actin related protein 2/3 domain, enolase, malate dehydrogenase and serine protease. These results suggest that the differential protein expression pattern could be exploited to identify putative virulence and pathogenicity factors in A. brassicae.