Soil salinity is one of the most significant abiotic stresses for crop growth and productivity worldwide. The United Nations Environment Programme estimated that approximately 20% of agricultural land and 50% of cropland in the world are under salinity stress (Flowers and Yeo, 1995). High concentration of salt in soil caused ion imbalance, oxidative damage, water deficit and nutrient deficiency, which led to molecular damage, growth retarded, and even death of plant. Salinity soil also spread widely in China. In the northeast of China, there is a more severe type of salinity soil that limiting the development of agriculture. Due to the presence of high exchange sodium percentage (ESP)(â‰§15) coupled with high pH (â‰§8.5) caused by CO32- and HCO3- accumulation after hydrolyzation of carbonate salts (Na2CO3 and NaHCO3) in parent material, thus this kind of soil was classified as alkali soil (George, 2005). Only a few plants species survive in such carbonate stress and they are distributed sparsely.
Recently, scientists have made various progresses on studying carbonate stress. Ghulam et al (2004) examined the effect of NaCl and Na2CO3 stress to link up with pH on plant growth of Arabidopsis thaliana and pearl millet. The toxic effect on the growth of Arabidopsis thaliana and pearl millet followed the order of Na2CO3> NaCl with adjusted pH as Na2CO3> NaCl only. Na2CO3 was found to be more toxic than NaCl even with pH equivalent to Na2CO3, suggesting that under Na2CO3 stress not only high pH retards plant growth but CO32- also has its own drastic effect on plant growth. Sustained irrigation of NaHCO3 decreased the growth (in terms of plant height) and yield of cotton (Choudhary et al., 2001). These studies showed that the effect of carbonate stress on plants is more severely than that of NaCl on plants; however, little was known on the molecular mechanism on the effect of carbonate stress on plants. Some studies on gene expression responding to carbonate stress have been reported. A cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. Two cDNA clones isolated from this library were respectively identified as mitochondrial ATP synthase 6 kD subunit gene (RMtATP6) and heat shock protein 90 gene (rHsp90). Transgenic tobacco over expressing the RMtATP6 gene and rHsp90 gene had greater tolerance to carbonate stress at the seedling stage than untransformed tobacco (Zhang et al., 2006; Liu et al., 2006).
In addition, the researches on special plants of alkali soil have been reported. The salt-tolerance mechanism of a halophyte, alkali grass (Puccinellia tenuiflora) was studied compared to a glycophyte, wheat with the results suggesting that alkali grass resists salt stress through high K+ and an endodermis barrier to Na+ (Peng et al., 2004). Leymus chinensis is a xerophilous grass and adapted to high pH (8.5~11.5) soda soils. Comparative EST profiles of leaf and root of L. chinensis under Na2CO3 stress led to the identification of 39 and 31 putative abiotic stress related genes from leaf and root cDNA library, respectively, these ESTs offered much information on some genes responding to carbonate stress (Jin et al., 2006).
Presently, a more powerful molecular biological technique, proteome, which is capable of giving insights into the quantity and quality of the final gene products, also the executor of biological function in the cell, i.e. the proteins, was developed rapidly. Proteomics is one of the high-throughput approaches that are being used to address biological function of plant by studying globally expressed proteins in a given tissue or subcellular organelle. However, only few proteomic studies on plants dealing with salt stress have been reported, which mainly focused on glycophytes, especially the model plant rice. In rice seedlings under salt stress, some stress-related proteins were identified by proteomic technique, such as auxin and salicylic acid response like protein (ASR1-like protein), ascorbate peroxidase (APX) and cafeoyl-CoA O-methyltransferase, UDP-glucose pyrophosphorylase (UDPG), cytochrome c oxidase subunit 6b-1, and glutamine synthetase, etc (Salekdeh et al., 2002; Abbasi and Komatsu, 2004; Yan et al., 2005; Kim et al., 2005). In another proteomic analysis targeted on pea (Pisum sativum) root under salt stress, pathogenesis-related proteins (PR10 proteins) were first identified as potential roles in salinity stress responses (Nat et al., 2004). It was hypothesized that glycophytes have most salt tolerance genes of halophytes, and use similar regulatory pathways and salt tolerance effectors (Zhu, 2000). To identify salt tolerance mechanisms of halophytes, a proteomic analysis was performed with a halophyte, Suaeda Aegyptiaca. 27 proteins were identified in Suaeda Aegyptiaca leaves treated with different salt levels. Among these proteins, cyanas is involved in cyanide detoxification, which is a new mechanism related to salt tolerance (Hossein et al., 2006). This was the first report on proteome patterns of a halophyte plant and its response to salt treatments, which offered significant meanings on mechanisms of plant to salt tolerance.
In this study, we investigated the protein patterns of a halophyte, C. virgata (Swartz) responding to carbonate stress by two-dimensional electrophoresis (2-DE) and MS analysis. C. virgata, also known as feather finger grass, is an annual C4, herbaceous graminoid native to warm temperate regions worldwide (Prendergast et al., 1976; Hickman, 1993). It is one of the few species that can grow naturally on the alkali soils in Northeast Plain of China. As its significant alkalinity tolerance and high productivity of biomass, C. virgata was regarded as one of the dominant species during succession and meliorating of alkali grassland. To our knowledge, this is the first study on proteomics of a halophyte response to carbonate stress.
1 Results and Discussion
1.1 Characterization of C. virgata tolerance to carbonate stress
The appearance of rice and C. virgata seedlings treated by 80 mM NaHCO3 for 3 days was shown in Figure 1. Compared with controls, the growth of rice seedlings were injured and retarded by sodium bicarbonate obviously in both shoots and roots. Conversely, the C. virgata seedlings were not damaged so severely by sodium bicarbonate compared with controls, except for slight reduction of the root length. After treated with 80 mM NaHCO3, the dry weight of roots of rice and C. virgata seedlings were examined (Figure 2A-B). The dry weights of both shoots and roots of stressed rice were lower than that of controls. This trend increased with the prolonging of the stress time. The dry weights of shoots and roots of rice, declined after 4 days and 2 days of treatment, respectively, which indicated that the seedlings began to die and the tissue began to decompose. This phenomenon was remarkable in root than in shoot, likely because the root is the first organ of plants to sense NaHCO3 stress, and the inhibition effect of NaHCO3 stress on rice roots growth is directly and more significant than that of shoots. Comparing with rice, the dry weights of shoots and root of C. virgata after treated by 80 mM NaHCO3 were not changed significantly. During the early stages of stress (within 3 days after stress for shoots and within 2 days after stress for roots), the dry weights of shoots and roots were even higher than that of controls, respectively, which indicated that as a halophyte, C. virgata is more tolerant to carbonate stress than rice.
1.2 Proteome analysis of C. virgata leaf under carbonate stress
2-week-old C. virgata rice seedlings were treated with 80 mM NaHCO3 for different time periods (24 h, 48 h, 72 h). Total proteins in leaves were extracted and separated by 2-DE using pH 3~10 IPG strips in IEF. After 2-D gel separation and CBB staining, about 500 protein spots were reproducibly detected on gels. The representative 2-DE maps were shown in Figure 3. 56 proteins showed significant and producible changes between control and treated samples until 72 h. 45 of them were up-regulated (spots 1-5, 7-12, 14-15, 17-22, 24-27, 29, 31-35, 37, 39-52, 55), 10 were down-regulated (spots 6, 16, 23, 28, 30, 38, 53-54, 56) (Figure 3A-B). Two frame regions in Figure 3 were enlarged in Figure 4. The relative abundance of protein spots, i.e, the percentage volumes in stress samples versus the percentage volumes in control samples, at 72 h point were shown in Figure 5. In order to confirm the creditability of the up-regulated and down-regulated of proteins showed in Figure 5, we analyzed the mRNA transcript level of some proteins corresponding to genes under 80 mM NaHCO3 stress using Northern blot (Figure 6). The cDNA fragment corresponding to each protein (spots 2, 5, 6, 7, 12, 14, 21) was amplified by RT-PCR as described in materials and methods. As shown in Figure 6, the genes expression of spot 2 and spot 14 was increased from 6 to 48 h. The spot 21 gene expression was induced strongly from 6 to 24 h, and decreased a little at 48 h. The mRNA accumulation of genes of spot 5, 7, and 12 decreased at 6 and 12 h, and increased maximally at 24 and 48 h. The spot 6 gene expression was not induced by NaHCO3 until 24 h. The results of Northern blot were almost consistent with that of proteomic analysis in a way.
Figure 3 2-D gel analysis of proteins extracted from leaves of C. virgata (Swartz)
Figure 4 Time course changes of leaf proteins of Chloris virgata (swartz.)
Figure 5 Relative abundance of leaf protein spots after 72 h of NaHCO3 treatment
Figure 6 Accumulation of novel NaHCO3 stress responsive genes transcript after exposure to NaHCO3 stress
1.3 Identification and classification of carbonate stress responsive proteins
Thirty protein spots with relatively high abundance were analyzed by MALDI-TOF MS and/or TOF-TOF MS. Seven of them with low match scores and not exceeded the significant threshold were regarded as failed match. Twenty-three protein spots identified with high probability were shown in Table 1. These proteins were classified to six categories according to their potential biological function in the cell, i.e., photosynthesis, metabolism, protease, transcriptional and translational factors, unknown proteins and other proteins.
1.3.1 Photosynthesis related proteins
As C4 metabolism species, 9 out of 23 proteins (39%) identified as carbonate stress responsive proteins are submitted to photosynthesis, indicating that regulation of photosynthesis may play important role on the C. virgata alkalinity tolerance mechanisms.
A symbol protein of C4 metabolism plants, phosphor- enolpyruvate carboxykinases (PEPCK) was identified and was up-regulated in two spots (spots 5, 7) after treatment of 80 mM NaHCO3 for 72 h. C4 plants have been classified as NADP-ME, NAD-ME, and PEPCK subtypes, according to the major decarboxylase involved in the decarboxylation of C4 acids in the bundle sheath cells (Gutierrez et al., 1974; Hatch et al., 1975). C. virgata belongs to the PEPCK subtype (Prendergast et al., 1976). In this subtype, phosphoenolpyruvate carboxykinase catalyze the nucleotide-dependent carboxylation of PEP to produce oxaloacetate (OAA) in reversible fashion as shown in equation (1).
CO2+ADP+PEP PEPCK OAA+ATP (1)
PEPCK-type C4 species mainly use aspartate as the CO2 donor and decarboxylate the OAA formed via the following reaction (2).
OAA+ATP PEPCK CO2+ADP+PEP (2)
Besides the important role in C4 photosynthesis, it was suggested that PEPCK might play a role in pH regulation in tissues active in the metabolism of nitrogen (Robert et al., 2001). In another report it was hypothesized that PEPCK involved in amino acids metabolism associated storage proteins (Walker et al., 1999). Three protein spots (spots 20, 21, 22) were identified as photosystem â…¡ oxygen evolving complex (OEC) protein 1 precursors, which share similar Mr but different pI values, indicating that they might be isoforms of PSII OEC protein 1 precursor. The OEC is localized on the lumenal side of PSII. It consists of the highly reactive Mn4+ cluster shielded by three extrinsics proteins. Ca2+ and Cl- also provide an optimal ionic environment for water oxidation (Schmidt and Mishkind, 1983). The OEC proteins are also named as the extrinsic 33 kD, 24 kD and 16 kD proteins, according to their apparent molecular masses of 34-33, 24-23 and 18-16 kDa (Bennett, 1981; Chia et al., 1986; Jensen et al., 1986). OEC-33 kD (OEC-33), believed to stabilize the Mn cluster of PSII (Barkan et al., 1986; Gollmer and Apel, 1983), two smaller proteins, OEC-24 kDa and OEC-16 kDa appear to play an auxiliary role in calcium and chloride binding (Tobin, 1983; Williams et al., 1986; Galfri and Milstein, 1981; Bricker et al., 1985). PSII OEC protein was up-regulated after cold stress (Cui et al., 2005). In our research, all the three PSII OEC protein 1 precursor were up-regulated by 80 mM NaHCO3, indicating that PSII OEC protein 1 precursor may play functional role during carbonate stress.
RubisCO is the key, first enzyme of the CO2 assimilation pathway. RubisCO large subunit is a key element of the RubisCO complex, a hexadecamer composed of eight RubisCO large subunits and eight RubisCO small subunits. RubisCO large subunit is the most abundance protein in plant chloroplast, with a molecular weight about 55 kD. In our research, however, the appearance of RubisCO large subunit (spot 23) was about 24.3 kD, with the protein identity is 100%. It is obviously that the RubisCO large subunit was degradation. This phenomenon has been observed in previous study (Norimoto et al., 2002; Zhao et al., 2005; Ferreira et al., 2000). After the lysates of chloroplasts isolated from naturally senescing wheat leaves incubated in darkness, the 44 kDa fragment of the large subunit of RubisCO, was found by immunoblotting with the specific antibodies (Norimoto et al., 2002). Other study indicated that RubisCO degradation is likely generated in vivo rather than by in vitro treatment (Zhao et al., 2005; Ferreira et al., 2000). The RubisCO large subunit is up-regulated by cold stress (Yan et al., 2005). In our research, the relative abundance of protein was slightly down-regulated by carbonate-salinity stress.
Also there are three other photosynthesis involved proteins responsive to carbonate stress, i.e, a down-regulated photosystem II oxygen-evolving complex protein 2 (fragment) (spot 28), an up-regulated putative photosystem I reaction center subunit â…¡ chloroplast precursor (Photosystem I 20 kDa subunit) (spot 47) and an up-regulated oxygen- evolving complex precursor (spot 48).
1.3.2 Metabolism associated proteins
5 of 23 spots were identified as proteins functionally related to carbohydrate and nitrogen metabolism. Spot 2 is an important enzyme for methionine metabolism, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (EC 22.214.171.124). Proteomic analysis of embryo axes tissue of hybrid maize seeds under cold germinated temperature showed that this protein was 2.55-fold up-regulated compared to control (Kollipara et al., 2002). In Pseudomonas aeruginosa, the methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase gene was 20-fold up-regulated by quorum sensing at transcriptional level (Victoria et al., 2002). In present research, methyltetrahydropteroy- ltriglutamate-homocysteine S-methyltransferase was 2.0-fold up-regulated by carbonate stress. Spot 12 is an up-regulated aspartate amino transferase. During osmotic and drought stressed, aspartate amino transferase activity of rice root and shoot was decreased (Pandey et al., 2004). Spot 13 and 14 were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is the key enzyme involved in glycolysis cycle. It has been suggested recently that Arabidopsis GAPDH has roles outside of its catalysis as part of glycolysis, while in other system this includes that of mediating reactive oxygen species (ROS) signalling (Hancock et al., 2005). The expression profiling of GAPDH was significantly regulated by oxidative, heavy metal and salt stress (Sweetlove et al., 2002; Jeong et al., 2001; Wang et al., 2006). Our results also consisted with that of the previous study (Wang et al., 2006). The spot 13 and 14 are GAPDH with similar Mr and pI indicating that they might be isoforms of GAPDH. The spot 13 only appeared in the control and treatment of 24 h and was 1.5-fold up-regulated by 80 mM NaHCO3 stress. Maybe this isoform only play its roles during the early growing and/or carbonate-salinity stress stage.
UDPase is a crucial enzyme involved in carbohydrate anabolism. It catalyzes the glucose-1-phospholate into UDP-glucose. UDP-glucose is directly used for cellulose synthesis (Amor et al., 1995). UDPase is involved in a flow of event in biosynthesis of cell wall components. In present study, UDPase was 2.6-fold up-regulated by carbonate stress, which suggested the accumulation of UDPase may fasten the cell wall biosynthesis, leading to the intensity of the defense effect on the cell wall. In rice, it was markedly reduced by salt stress (Yan et al., 2005). In Arabidopsis, UDPase protein was strongly induced by sucrose, light, cold stress and phosphate deficiency but reduced by drought and flooding (Ciereszko et al., 2001a; Ciereszko et al., 2001b). In present research, UDPase protein (spot 15) 2.6-fold up-regulated by carbonate stress in C. virgata, suggesting that as a halophyte, sodium bicarbonate stress might trigger its carbonate tolerance by enhancement of anabolism products accumulation and fasten of the cell wall biosynthesis.
Proteolytic processes triggered by proteases take place inside cells are necessary for cell viability regulation. Spot 6 is a putative FtsH-like protein Pftf precursor and act as a Zn2+-dependent metalloprotease. Pftf is an ATP-dependent metalloprotease that is encoded by nuclear genes and post-translational imported into the membrane of chloroplasts by specific targeting sequences (Adam, 2000). In plant, it is light-inducible and can degrade the unassembled Rieske Fe-S proteins and D1 proteins of photosystem â…¡ (Lindahl et al., 2001). AtFtsH6 is involved in the degradation of the light-harvesting complex â…¡ during high-light acclimation and senescence (Haussuhl et al., 2001). Also FtsH-like protein Pftf precursor was up-regulated by low temperature in rice (Zhao et al., 2005). After 72 h treatment of 80 mM NaHCO3, the relative abundance of protein was down-regulated, due to the essential to maintaining the proteins of photosystem â…¡ after carbonate stress.
1.3.4 Transcriptional factors
Gene expression can be regulated at transcriptional, post-transcriptional, translational, and post-translational levels. Many studies of the transcriptional activation of gene expression have reported that transcription factors play a major role in stress response pathways (Busch et al., 2005; Henriksson and Nordin, 2005). In this study, three up-regulated proteins involved in transcriptional factors, include pentatricopeptide (PPR) repeat-containing protein-like (spot 25), transcription initiation factor IIF, alpha subunit family protein (spot 27) and methyl-CpG binding protein-like (spot 18).
PPR is a large gene family discovered by Small and Peters (2000). It is a characteristic repeated motif consisted of tandem 35 amino acids. Functionally characterized proteins of this family are all implicated in RNA-processing events in both mitochondria and chloroplasts. In mitochondria, for example, a PPR gene restores fertility to cytoplasmic male-sterile (CMS) of P. parodii through control of related genes (Bentolila et al., 2002). Also PPR might play roles in cold stress defense pathways (Kollipara et al., 2002). In our research, pentatri- copeptide (PPR) repeat-containing protein-like was 1.65-fold up-regulated after treatment of 80 mM NaHCO3 for 72 h indicating that it might play roles in RNA processing in response to carbonate stress.
In Schizosaccharomyces pombe, one of the subunits of transcription initiation factor (TFIIF), Tfg3 was reported to be involved in transcriptional regulation under high temperature (Kimura and Ishihama, 2004). In our research, the up-regulation of a transcription initiation factor IIF, alpha subunit family protein subjected to carbonate stress suggest that TFIIF α might also be involved in transcriptional regulation of the response of plant to carbonate stress.
DNA methylation, one of the most abundant epigenetic modifications in higher plants and animals, plays an important role in regulating development and developmental processes (Meehan, 2003; Finnegan et al., 2000). In Arabidopsis, ten members of the DNA methylational gene family encoding methyl-CpG- binding domain proteins are transcriptional active and at least one, AtMBD11, is crucial for normal development (Berg et al., 2003). In our study, a methyl-CpG binding protein-like was 2-fold up-regulated by carbonate stress indicating that it might play important roles for maintaining normal development of C. virgata under carbonate stress.
1.3.5 Other proteins
Other proteins are ribosomal protein L18P/L5E (spot 16) and PREDICTED OJ1767_D02.15-2 gene product (spot 19). Ribosomal protein L18P/L5E with function of structural constituent of ribosome is a family includes L18 from bacteria and L5 from eukaryotes. The ribosomal 5S RNA is the only known rRNA species to bind a ribosomal protein before its assembly into the ribosomal subunits (Deshmukh et al., 1993). In Medicago truncula, it was found that three ESTs which encode a ribosomal protein L18P/L5E, respectively, were up-regulated at least 2-fold by Pseudomonas. fluovescens colonization of roots (Sanchez et al., 2005). Our research also suggested that ribosomal protein L18P/L5E is involved in the carbonate tolerance of C. virgata.
1.3.6 Unknown proteins
Unknown proteins include spot 29, 32 and 36. Potential functions of these proteins were predicted by software, ProtFun 2.2 Server (http://www.cbs.dtu.dk/ services/ProtFun/). Protein 29 may related with amino biosynthesis; protein 32 may participates in process of positive regulation of angiogenesis and functions as (glutamate-ammonia-ligase) adenylyltransferase activity; protein 34 may play roles in lipid transporter activity. They also might be involved in the carbonate tolerance of C. virgata during sodium bicarbonate treatment.
2 Concluding Remarks
To our best knowledge, this is the first study on proteomics of a halophyte response to carbonate stress. Image analysis of protein spots showed 56 leaf proteins response to 80 mM NaHCO3 treatment significantly. The majority of carbonated stress-induced changes were up-regulation in abundance. 23 out of 56 protein spots were identified by MS analysis. A large part (9 out of 23) of these proteins is involved in photosynthesis suggesting that as a C4 halophyte, C. virgata regulates its carbonate tolerance by adjusting the photosynthesis process. Photosynthesis related proteins may play important roles during carbonate stress. Proteins related to carbohydrate and nitrogen metabolism are also important in carbonate tolerance of C. virgata, especially a protein that mediate reactive oxygen species (ROS) signaling, GAPDH. A chloroplast putative FtsH-like protein Pftf precursor was identified a novel salt-responsive protein in our research and essential to maintaining the proteins of photosystem â…¡ after carbonate stress. Also transcription factors may play important roles in carbonate stress response pathways. The proteins identified in present study represent only a small part of the C. virgata proteome response to carbonate stress, the remaining proteins still need to be identified. Meanwhile, further functional analysis of these proteins may be necessary in understanding of carbonate tolerance mechanisms of plant. This study will give new insight into alkalinity stress response in plant.
3 Materials and Methods
3.1 Plant materials
Seeds of C. virgata were collected around the alkali spots of Songnen grassland, Heilongjiang province, China. The seeds of C. virgata and rice (O. sativa L. cv. Nipponbare) were surface sterilized 70% ethanol for 1 min and 10% NaClO for 10 min, and followed by a thorough washing in distilled water. The seeds were soaked in distilled water and germinated. The cultural condition was controlled at a 25â„ƒ, photoperiod 16 h, relative humidity 60%, and a Photon flux density 220 mM/m26S. After 1 week, the seedlings were cultured in half-strength Hoagland’s solution for another week. For dry mass determination, the 2-week-old seedlings were treated with 80 mM NaHCO3 for 1, 2, 3, 4, 5 and 6 days, with distilled water as controls. The roots and shoots were dried at 80â„ƒ for 48 h in an oven. Each 10 rice plants and 200 C. virgata plants were taken as one sample, respectively. Each experiment was repeated three times.
For proteomic and northern blot analysis, the 2-week-old C. virgata seedlings were treated with 80 mM NaHCO3. Differently, for proteomic analysis, the seedlings were treated for 1 d, 2 d and 3 d, respectively; and for northern blot analysis, the seedlings were treated for 6 h, 12 h, 24 h and 48 h, respectively. The seedlings treated with distilled water were as the control. After treatment, the leaves were harvested. For each treatment, there were four replicates.
3.2 Protein extraction
Plant tissues added 1% PVPP were grinded using liquid nitrogen and suspended in ice cold 10% (w/v) TCA in acetone containing 0.07% (w/v) DTT, incubated at -20â„ƒ overnight and centrifuged for 15 min at 35 000 g. The pellets were resuspended in ice cold acetone added 0.07% (w/v) DTT, incubated at -20â„ƒ for 1 h and centrifuged for 15 min at 16 000 g. This step was repeated at least three times and the pellets were lyophilized. The resulting powder was solubilized in lysis buffer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2.5% (v/v) pH 3~10 IPG buffer, 1% (w/v) DTT, 1 mM PMSF) for 1 h at 36 â„ƒ followed by centrifugation for 15 min at 35 000 g. The supernatant containing proteins was transferred into fresh tubes. Protein concentration was determined using modified Bradford assay with BSA as standard. The protein samples were stored at -80â„ƒ.
3.3 Two-dimensional electrophoresis (2-DE)
For 2-DE analysis, 450 µg proteins supplied with rehydration buffer (8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) pH 3~10 IPG buffer, 20 mM DTT) to a total volume of 250 µL were loaded onto 13 cm dry IEF strips, pH 3~10 linear gradient (Amersham Biosciences, Uppsala, Sweden). For IEF, the IPGphor system (Amersham Biosciences, Uppsala, Sweden) was used. The program was as follows: 30 V for 12 h, 200 V for 1 h, 1 000 V for 1 h, 8 000 V gradient for 0.5 h and 8000 V for 5 h. The maximal current was 50 µA per strip. The gel strips were equilibrated in 5 mL equilibration buffer (50 mM Tris-HCl buffer, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT and 0.002% (w/v) bromophenol blue) for 15 min at room temperature followed by another equilibration step only with the replacement of 2.5 % (w/v) iodoacetamide to 1% (w/v) DTT. SDS-PAGE was performed with 12.5% gels using the SE 600 system (Amersham Biosciences, Uppsala, Sweden). The gels were run at 15 mA per gel for about 6 h each. The protein gels were stained with Coomassie Brilliant blue R-250. At least three replicates were performed for each sample.
3.4 Image analysis
Gels were scanned using a GS-700 densitometer (Bio-Rad) at a resolution of 300 dpi. Image treatment, spot detection, protein quantification, gel matching and spots filtering were carried out using the Image master 3.01 software (Amersham Pharmacia Biotech). The abundance of each protein spot was estimated by the percent of volume (% Vol). Only those protein spots that abundance changed above 1.5 fold between control gels and treated gels in at least one stage of the carbonate stress cycle were selected for MS analysis. The molecular masses of protein on gels were determined by co-electrophoresis of standard protein markers and pI of the proteins were determined by migration of the protein spots on strips.
3.5 Protein identification and database search
Protein spots were excised from polyacrylamide gels that had been stained with Coomassie Brilliant Blue R-250 and washed three times with ultra pure water. The protein spot gels were destained twice with 50 mM NH4HCO3 in 50% acetonitrile, 20 minutes for each time. Then the gels were dried with 100% acetonitrile and digested overnight at 37â„ƒ with trypsin. The peptide were extracted twice or thrice with 0.1%TFA in 50% acetonitrile. Extracts were lyophiliaed with N2 and prepared for MS analysis. Protein identification was accomplished by MALDI-TOF-MS combined with MALDI-TOF-TOF with a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). For MALDI-TOF-MS, PMFs obtained with a tolerance of +0.3 kD and one missed cleavage site. Significance level of peptide mass fingerprint result was set at 5%. TOF-TOF-MS was performed when PMFs obtained from a particular spot did not provide sufficient information for protein identification. Data were analyzed using GPS Explorer software (Applied Biosystem) and MASCOT software (Matrix Science, London, UK). NCBInr and Viridiplantae as taxonomi were selected as the database and taxonomy category, respectively.
3.6 Northern blot analysis
For Northern blot analysis, plant total RNA was isolated using Trizol reagent (Invitrogen) according to manufacturer’s instructions. 10 μg plant total RNA was separated on denaturing formaldehyde 1.0% agarose gel, and blotted onto a nylon membrane. According to the information of identified proteins of spots 2, 5, 6, 7, 12, 14 and 21 accomplished by MALDI-TOF-MS, the conserve domain of each protein were confirmed. The mix-primers were designed according to the conserve domain of each protein. The cDNA corresponding to each protein was amplified using the mix-primers by RT-PCR, and then each cDNA fragment was labelled by digoxigenin (DIG) as a probe for hybridiazation. Hybridization signals were detected with CDP-StarTM (Amersham Pharmacia).
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