Two-Dimensional Electrophoretic Analysis of Soluble Leaf Proteins of a Salt-sensitive(Triticum aestivum)and a Salt-tolerant(T.durum)Cultivar in Response to NaCl Stress

In this research, 3-day-old etiolated wheat seedlings of Triticum aestivum L. cv. Ceyhan-99 (salt-sensitive) and T. durumDesf. cv. Firat-93 (salt-tolerant) were grown in control and salt (150 mmol/L NaCI) treatments at a 15/25℃ temperatureregime in the light for 12 days. Soluble proteins extracted from the first leaf tissues of two cultivars were analyzed by two-dimensional (2-D) electrophoresis in order to detect NaCl-induced changes. The soluble leaf protein profiles of cultivarswere observed to be similar. However, quantitative differences in 74 proteins were detected in the salt treatment group,compared to the control. Among the 74 protein spots, 14 were common for two cultivars. As a result of NaCl treatment, twolow-molecular-weight (LMW) proteins (28.9 and 30.0 kDa) and one intermediate-molecular-weight (IMW) protein (44.3 kDa)in cv. Ceyhan-99 and six LMW proteins (18.6, 19.4, 25.7, 25.9, 26 and 27.6 kDa) in cv. Firat-93 were newly synthesized. Thenewly synthesized proteins were specific to each cultivar. In the Firat-93 cultivar, four proteins with LMW (24.8-27.9 kDa)were completely lost in NaCl treatment. Moreover, these four protein spots were not observed in both protein profiles ofcv. Ceyhan-99. Most of these proteins were in acidic character (pi<6.0-6.9) and low molecular weight (<31.6 kDa). It issuggested that the newly synthesized or completely lost LMW proteins may be important for cultivars differing in sensitivitytowards NaCl.     

Protein Changes in Response to Pyrene Stress in Maize （Zea mays L.） Leaves

Phytoremedlation is a relatively new approach to remove polycyclic aromatic hydrocarbons （PAHs） from the environment. When plants are grown under pyrene treatment, they respond by synthesizing a set of protective proteins. To learn more about protein changes in response to pyrene treatment, we extracted total proteins from the leaves of maize （Zea mays L.） 1 week after pyrene treatment. The proteins extracted were separated with twodimensional gel electrophoresis. In total, approximately 54 protein spots were found by comparing gels from treated and control groups. According to the Isoelectric point, molecular weight, and abundance of these protein spots, 20 pyrene-lnduced proteins were found to have changed abundance. Of these, 15 protein spots were Increased and five protein spots were newly appeared in pyrene-treated plant leaves. Six model upregulated protein spots of different molecular weights were excised from the gels and subjected to trypsin digestion followed by peptide separation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptlde masses were used to search the matrix-science database for protein Identification. Two of the proteins were Identified on the basis of the homology of their peptide profiles with existing protein sequences as pyruvate orthophosphate diklnase and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunlt. These proteins are Involved in the regulation of carbohydrate and energy metabolism. The present study gives new Insights into the pyrene stress response In maize leaves and demonstrates the power of the proteomlc approach in phytoremedlation of PAHs.     

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase （spot 27） participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress （spots 8, 115, 118, 125, 130） and signal transduction （spots 7, 100, 108）. According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm （spots 54, 61, 73） were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo （spots 27, 62, 122） were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

Proteomic Alterations of Antarctic Ice Microalga Chlamydomonas sp. Under Low-Temperature Stress

Antarctic ice microalga can survive and thrive in cold channels or pores in the Antarctic ice layer. In order to understand the adaptive mechanisms to low temperature, in the present study we compared two-dimensional polyacrylamide gel electrophoresis （2-DE） profiles of normal and low temperature-stressed Antarctic ice microalga Chlamydomonas sp. cells. In addition, new protein spots induced by low temperature were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. Well-resolved and reproducible 2-DE patterns of both normal and low temperature-stressed cells were acquired. A total of 626 spots was detected in control cells and 652 spots were detected in the corresponding low temperature-stressed cells. A total of 598 spots was matched between normal and stressed cells. Two newly synthesized proteins （a and b） in low temperature-stressed cells were characterized. Protein spot A （53 kDa, pl 6.0） was similar to isopropylmalate/homocitrate/citramalate synthases, which act in the transport and metabolism of amino acids. Protein spot b （25 kDa, pl 8.0） was related to glutathione S-transferase, which functions as a scavenger of active oxygen, free radicals, and noxious metabolites. The present study is valuable for the application of ice microalgae, establishing an ice microalga Chlamydomonas sp. proteome database, and screening molecular biomarkers for further studies.     

Salinity Stress in Roots of Contrasting BarleyGenotypes Reveals Time-Distinct and Genotype-Specific Patterns for Defined Proteins

Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence,particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance onsaline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are wellcharacterized. A proteomic analysis of the roots of two contrasting cultivars （cv. Steptoe and cv. Morex） is presented.Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150mM NaCI. The root proteome was analyzedbased on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins wereidentified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysisgrouped these into five clusters based on similarity of expression profile. The rank product method was applied to sta-tistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinitytolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerousas-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis weredetected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms andmembrane stability via fine tuning of phytohormone and secondary metabolism in the root.     

Promotion by 5-Aminolevulinic Acid of Germination of Pakchoi （Brassica campestris ssp. chinensis var. communis Tsen et Lee） Seeds Under Salt Stress

The seed germination and seedling growth of pakchoi （Brassica campestris ssp. chinensis var. communis Tsen et Lee cv. Hanxiao） were not significantly inhibited until the concentration of NaCl was increased to 150 mmol/L. Treatment of pakchoi seeds with exogenous 5-aminolevulinic acid （ALA）, at concentrations ranging from 0.01 to 10.00 mg/L, promoted seed germination when seeds were stressed by salinity, whereas levulinic acid （LA）, an inhibitor of ALA dehydrase, significantly inhibited seed germination and seedling growth, suggesting that metabolism of ALA into porphyrin compounds was necessary for seed germination and seedling growth. Determination of respiratory rate during seed germination showed that ALA increased seed respiration under both normal conditions and salt stress. Furthermore, salt stress decreased levels of endogenous ALA, as well as heme, in etiolated seedlings. More salt-tolerant cultivars of pakchoi contained higher relative levels of endogenous ALA and heme under conditions of salt stress. These results indicate that salt stress may inhibit the biosynthesis of endogenous ALA and then heme, which is necessary for seed germination, and treatment of seeds with exogenous ALA prior to germination may be associated with the biosynthesis of heme.     

Identification of differentially expressed proteins in poplar leaves induced by Marssonina brunnea f. Sp. Multigermtubi

Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. sp. multigermtubi. To date, little is known about the molecular mechanism of poplar （M. brunnea） interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone ＂NL895＂ （P. euramericana CL＂NL895＂）, which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study. We used two-dimensional gel electrophoresis （2-DE） and mass spectrometry （MS） to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. sp. multigermtubi. Proteins extracted from poplar leaves at 0, 12, 24, 48, and 72 h after pathogen-inoculation were separated by 2-DE, About 500 reproducible protein spots were detected, of which 40 protein spots displayed differential expression in levels and were subjected to Matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS） followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and unclassified proteins.     

Small heat shock protein responses in leaf tissues of wheat cultivars with different heat susceptibility

The effect of heat stress on soluble proteins extracted from leaf tissues of bread (Triticum aestivum cv. Gönen-98, tolerant; cv. Cumhuriyet-75, susceptible; genome ABD) and durum (Triticum durum cv. Ege-88, tolerant; cv. Ankara-98, susceptible; genome AB) wheat cultivars differing in sensitivity to high temperature was examined by two-dimensional gel electrophoresis. At acclimation (37°C) and acclimation→high temperature (37°C→50°C) treatments compared to control (25°C), evaluation of gels revealed 31 proteins to be differentially expressed in first leaves as a result of heat stress in heat-susceptible and heat-tolerant cultivars of bread and durum wheats. All of the increased or decreased proteins in amount, newly synthesized and/or disappeared were in low-molecular-weight (LMW, 16.1–24.0 kDa) and generally acidic character (pI 4.8–6.9). The responses of the four cultivars were compared: Twenty-two of 31 proteins were detected as newly synthesized LMW heat shock proteins (LMW HSPs = small HSPs). The number of these sHSPs was different in cultivars which have the same genome. In addition, the number of the sHSPs in heat-tolerant cultivars was higher than in heat-susceptible cultivars. Some of the sHSPs were specific to cultivar. Most of the sHSPs synthesized at 37°C were also detected at 37°C→50°C treatment. It is suggested that sHSPs have special importance in two points: Firstly, sHSPs in cultivars showed abundance and diversity. Secondly, these proteins may play an important role in the acquiring of thermal tolerance.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

The alteration of mRNA expression of SOD and GPX genes,and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles

Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L⁻¹) and ZnO-NPs (0, 15 and 30 mg L⁻¹). Treatments with NaCl at both 3 and 6 g L⁻¹ suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS–PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.     

Differential expression of LEA proteins in two genotypes of mulberry under salinity

The relative water content (RWC), cell membrane integrity, protein pattern and the expression of late embryogenesis abundant proteins (LEA; group 1, 2, 3 and 4) under different levels of salt stress (0, 1.0, 1.5 and 2.0 % NaCl) were investigated in mulberry (Morus alba L.) cultivars (S1 and ATP) with contrasting salt tolerance. RWC and membrane integrity decreased with increase in NaCl concentration more in cv. ATP than in cv. S1. SDS-PAGE protein profile of mulberry leaves after the NaCl treatments showed a significant increase in 35, 41, 45 and 70 kDa proteins and significant decrease in 14.3, 18, 23, 28, 30, 42, 47 and 65 kDa proteins. Exposure of plants to NaCl resulted in higher accumulation of LEA proteins in S1 than ATP. The maximum content of LEA (group 3 and 4) was detected in S1 at 2.0 % NaCl, which correlates with its salt tolerance.     

Proteome analysis of potato under salt stress

Because salt stress is a major abiotic source of stress on potato crops, the molecular mechanism of the response of potato plants to salt stress was examined. On exposure to salt, the salt-sensitive cultivar Concord showed a greater reduction in shoot and root length than did the salt-tolerant cultivar Kennebec. For both cultivars, the reduction in the length of shoots was more severe than that of the roots. Salt exposure increased the content of free proline and total soluble sugars in shoots of Kennebec; these remained unchanged in Concord. Proteins extracted from shoots of both cultivars exposed to 90 mM NaCl were separated by two-dimensional polyacrylamide gel electrophoresis: 322 and 305 proteins were detected in shoots of Kennebec and Concord, respectively. Of these, 47 proteins were differentially expressed under NaCl treatment in shoot of both cultivars. Among the differentially expressed proteins, photosynthesis- and protein-synthesis-related proteins were drastically down-regulated, whereas osmotine-like proteins, TSI-1 protein, heat-shock proteins, protein inhibitors, calreticulin, and five novel proteins were markedly up-regulated. These results suggest that up-regulation of defense-associated proteins may confer relative salt tolerance to potato plants.     

Accumulation of a High Molecular Weight Protein during Cold Hardening of Wheat (Triticum aestivum L.)

Electrophoretic patterns of soluble protein fractions from cold-tolerantwinter wheats (Triticum aestivum L. cv. Frederick and cv. Norstar)and cold-sensitive spring wheat (T. aestiaum L. cv. Glenlea)were analysed in hardened and unhardened plants. One and two-dimensionalgel electrophoresis analysis reveals that cold hardening conditionsinduce changes in the soluble protein patterns. The most importantis the accumulation of a high molecular weight protein in therange of 200 kDa. This protein accumulated at higher concentrationin cold-tolerant cultivars compared to the coldsensitive onesuggesting a correlation between the degree of freezing toleranceand the accumulation of this specific protein. In addition,the intensity of three protein bands (mol wt 48, 47 and 42 kDa)increased while that of five others (mol wt 93, 89, 80, 67 and63 kDa) decreased during hardening. These changes occured inthe three cultivars suggesting that they are part of the metabolicadjustments in response to low temperature rather than a specificchange associated with the development of cold hardiness. (Received April 23, 1987; Accepted June 5, 1987)     

Low temperature treatment at the young microspore stage induces protein changes in rice anthers

Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (<20 degrees C) at the young microspore stage induce severe pollen sterility and thus large grain yield reductions. To investigate this phenomenon, anther proteins at the early stages of microspore development, with or without cold treatment at 12 degrees C, were extracted, separated by two-dimensional gel electrophoresis, and compared. The cold-sensitive cultivar Doongara and the relatively cold-tolerant cultivar HSC55 were used. The abundance of 37 anther proteins was changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv. Doongara. Among them, one protein was newly induced, 32 protein spots were up-regulated, and four protein spots were down-regulated. Of these 37 protein spots, we identified two anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were down-regulated and a cystine synthase, a beta-6 subunit of the 20 S proteasome, an H protein of the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein homologue, a putative 6-phosphogluconolactonase, a putative adenylate kinase, a putative cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase, and a monodehydroascorbate reductase that were up-regulated. Identification of these proteins is available upon request. Accumulation of these proteins did not vary greatly after cold treatment in panicles of cv. Doongara or in the anthers of the cv. HSC55. The newly induced protein named Oryza sativa cold-induced anther protein (OsCIA) was identified as an unknown protein. The OsCIA protein was detected in panicles, leaves, and seedling tissues under normal growth conditions. Quantitative real time RT-PCR analysis of OsCIA mRNA expression showed no significant change between low temperature-treated and untreated plants. A possible regulatory role for the newly induced protein is proposed.     

Proteomic Study of Low-Temperature Responses in Strawberry Cultivars (Fragaria x ananassa) That Differ in Cold Tolerance

To gain insight into the molecular basis contributing to overwintering hardiness, a comprehensive proteomic analysis comparing crowns of octoploid strawberry (Fragaria × ananassa) cultivars that differ in freezing tolerance was conducted. Four cultivars were examined for freeze tolerance and the most cold-tolerant cultivar ('Jonsok') and least-tolerant cultivar ('Frida') were compared with a goal to reveal how freezing tolerance is achieved in this distinctive overwintering structure and to identify potential cold-tolerance-associated biomarkers. Supported by univariate and multivariate analysis, a total of 63 spots from two-dimensional electrophoresis analysis and 135 proteins from label-free quantitative proteomics were identified as significantly differentially expressed in crown tissue from the two strawberry cultivars exposed to 0-, 2-, and 42-d cold treatment. Proteins identified as cold-tolerance-associated included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis-related proteins, and flavonoid pathway proteins. A number of proteins were newly identified as associated with cold tolerance. Distinctive mechanisms for cold tolerance were characterized for two cultivars. In particular, the 'Frida' cold response emphasized proteins specific to flavonoid biosynthesis, while the more freezing-tolerant 'Jonsok' had a more comprehensive suite of known stress-responsive proteins including those involved in antioxidation, detoxification, and disease resistance. The molecular basis for 'Jonsok'-enhanced cold tolerance can be explained by the constitutive level of a number of proteins that provide a physiological stress-tolerant poise.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.