Technical improvement to 2D-PAGE of rice organelle membrane proteins

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.     

A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl cross-linking reagent tetrathionate : Polypeptides of the internal nuclear matrix

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis

High-resolution two-dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1,398 polypeptides (1,127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus-induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.     

Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella

Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats. CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6. Immunoblotting revealed most of the stained bands around pH 4.5-5.4. CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested. Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

苜蓿体细胞胚胎发生过程中DNA、RNA和蛋白质的合成动态

苜蓿(Medicago sativa L.)下胚轴切段产生的愈伤组织经2,4-D短时间诱导后,在无激素液体培养基中可形成大量体细胞胚胎。经2,4-D诱导后的愈伤组织在转入无激素培养基1天后,其DNA、RNA和蛋白质的合成即进入活跃合成状态,并在体细胞胚胎发育过程中保持逐步升高的趋势。在苜蓿体细胞胚胎发生过程中,有些蛋白质组分含量减少或消失,但绝大部分蛋白质组分的含量明显增加,并且有若干新蛋白的出现,其中24 KD和46 KD蛋白质为体细胞胚胎发生早期所特有。     

Quantitative changes in maize cytoplasmic proteins induced by aluminium

Three-day-old maize seedlings were subjected to 100 μM AlCl3 for 24 h. Cytoplasmic proteins were isolated from root tips, root base and from coleoptiles. After fractionation of cytoplasmic proteins on anion chromatography column Bio-Scale Q2 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to monitor Al-induced changes in polypeptide composition of particular fractions. Four (root) and 7 (coleoptile) fractions were eluted from the column with linear 0 - 1.0 M NaCl gradient. In fraction 1 of cytoplasmic proteins from root tips Al induced accumulation of polypeptide with molecular mass of 16 kD and simultaneous reduction of two polypeptides (67.5 and 60 kD). In fraction 1 isolated from mature zone of maize roots Al-induced accumulation of 22 kD polypeptide and reduction of 67.5, 60, and 14 kD polypeptides. Most pronounced changes were revealed in coleoptile. In three protein fractions increased accumulation of polypeptides with molecular mass of 14, 17.5, 20, 24.5, 28, 30, and 37.5 kD were observed. In the remaining three root or four coleoptile fractions of cytoplasmic proteins, no differences were found between Al-treated and control maize seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.     

蛋白质组学研究中的对角线电泳

经典的蛋白质组学研究方法包括IEF/SDS-PAGE双向电泳和质谱技术的联用,但由于IEF的一些不足,限制了其应用范围。对角线电泳是蛋白质组学研究中的一项特殊分离技术,由于其原理与IEF/SDS-PAGE不同,正逐渐成为蛋白质组学中电泳分离技术的重要补充,特别是在膜蛋白和蛋白质相互关系的研究中将起到重要作用。本文综述了对角线双向电泳技术的特点、发展和在蛋白质组学研究中的最新进展,比较了双向电泳和对角线电泳的优缺点,展望了对角线电泳在蛋白质组学研究中的应用前景。     

Oxidative stress in greater duckweed (Spirodela polyrhiza) caused by long-term NaCl exposure

The mechanisms of aquatic plant defense against salinity were studied by long-term exposure of Spirodela polyrhiza (greater duckweed) to NaCl. In this study, the effects of 200 mM NaCl on greater duckweed were evaluated after 6 and 12 days of treatment, while plant growth was measured every day. High concentration of NaCl caused an inhibition of plant growth, reduced in the content of photosynthetic pigments, increased lipid peroxidation, and enhanced the entire antioxidant defense. The responses of five antioxidant enzymes showed that ascorbate peroxidase, guaiacol peroxidase, and superoxide dismutase activities were the most enhanced after NaCl exposure, catalase moderately, and glutathione reductase least. The content of soluble proteins was decreased, while ascorbic acid was drastically increased. In NaCl-treated fronds, the appearance of two NaCl-induced polypeptides with apparent molecular weight of 16 and 21 kDa, as well as the accumulation of two polypeptides with molecular weights 18 and 27 kDa, were observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). NaCl also led to accumulation of the heat shock protein 70 (HSP70) and induced an isoform of the glutamine synthetase (GS1) expression. Our results suggest that in S. polyrhiza, different adaptive mechanisms are involved in counter balancing high doses of a particular toxicant (sodium chloride). The possible application of the examined biomarkers in ecotoxicological research is discussed.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Quantitative Changes in Maize Membrane Proteins Induced by Aluminium

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for monitoring Al-induced changes in polypeptide composition of membrane proteins isolated from 3-d-old maize seedlings subjected to aluminium stress. Analysis of peripheral membrane proteins isolated from maize root showed an Al-induced increase in accumulation of 14 polypeptides with apparent molecular mass from 10 to 135 kDa. Qualitative differences were found between peripheral membrane proteins isolated from root tip (increased accumulation of 4 polypeptides with M_r 42 000 – 135 000) and from root base (increased accumulation of 10 polypeptides with M_r 10 000 – 59 000). On the other hand, no Al-induced changes were observed in peripheral membrane proteins isolated from maize coleoptile and integral membrane proteins isolated either from root or coleoptile. These results indicate that peripheral membrane proteins undergo considerable changes during 24-h Al treatment while integral membrane proteins pattern is stable.     

Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography

The major storage proteins of maize (Zea mays L.) endosperm are located in protein bodies, and may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into two major classes and four minor classes of polypeptides. The two major classes (commonly known as zeins) have been separated previously into a large number of components by isoelectric focusing (IEF). Reversed-phase high performance liquid chromatography (HPLC) further separated the major classes into additional components, and gave distinctive peaks for each minor zein class. Some IEF bands produced two or more HPLC fractions, while some HPLC fractions produced two or more IEF bands. Apparently identical IEF bands from different inbreds may appear in different fractions after HPLC. Thus the total number of zeins revealed by separations based on apparent size (SDS-PAGE), net charge (IEF), and hydrophobicity (HPLC) is very large. Different laboratories have developed diverse nomenclatures which cause much confusion. A key is presented to provide a flexible and expandable nomenclature for this complex group of proteins.     

Salt-stress induced alterations in protein profile and protease activity in the mangrove Bruguiera parviflora

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mM) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mM NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.