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First published online November 26, 2008; 10.1104/pp.108.131144 Plant Physiology 149:916-928 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Identification of an Apoplastic Protein Involved in the Initial Phase of Salt Stress Response in Rice Root by Two-Dimensional Electrophoresis1,[C],[W],[OA]Institute of Molecular and Cell Biology, Hebei Normal University, Shijiazhuang 050016, China
The apoplast of plant cells, which carries out multiple functions in plant metabolism and signaling, is not only a barrier but also the linker between the environment and the protoplast. To investigate the role of apoplastic proteins in the salt stress response, 10-d-old rice (Oryza sativa) plants were treated with 200 mM NaCl for 1, 3, or 6 h, and the soluble apoplast proteins were extracted for differential analysis compared with untreated controls using two-dimensional electrophoresis. Ten protein spots that increased or decreased significantly in abundance were identified by mass spectrometry. These proteins included some well-known biotic and abiotic stress-related proteins. Among them, an apoplastic protein, with extracellular domain-like cysteine-rich motifs (DUF26), O. sativa root meander curling (OsRMC), has shown drastically increased abundance in response to salt stress during the initial phase. OsRMC RNA interference transgenic rice has been generated to assess the function of OsRMC in the salt stress response. The results show that knocking down the expression level of OsRMC in transgenic rice led to insensitive seed germination, enhanced growth inhibition, and improved salt stress tolerance to NaCl than in untransgenic plants. These results indicate that plant apoplastic proteins may have important roles in the plant salt stress response.
Salt stress is one of the most significant abiotic stresses and affects every aspect of plant physiology and metabolism. During salt stress, Na+ enters the cells and accumulates to a concentration that induces ionic and osmotic stress in plants. Plant cells respond and adapt to these adverse conditions through signaling networks (Lee et al., 2004  ). Understanding the signaling pathway of plant salt resistance is important for improving plant salt tolerance, especially for improving agricultural productivity in irrigated land.
To study the signaling network of plant salt adaptation, the most important thing is identification of the components involved. Much effort has been made to discover components or elements of signaling pathways involved in plant salt stress responses. Of all the molecular components and signaling pathways known so far, the best understood signaling pathway in salt ionic stress is the SOS pathway (Zhu, 2002
The apoplast is the portion of the plant cell outside the cell membrane. This region includes the cell walls and intercellular space of the plant (Dietz, 1997
The sequencing of many plant and animal genomes has revealed the fact that the regulation of a cell's biological activities mostly occurs at the level of protein degradation, interactions, and posttranslational modification. Proteomics is now emerging as a powerful tool for studying these protein dynamics, especially in plant stress responses (Salekdeh et al., 2002
Apoplastic Extracts Are Free of Cytosolic Contamination The vacuum infiltration method was used to obtain apoplastic fluid from the rice root. Because the procedures of vacuum infiltration and centrifugation could damage cells and contaminate apoplastic extracts with cytosolic ingredients, we used malate dehydrogenase (MDH) activity assay and immunobloting against tubulin to evaluate the quality of apoplastic extracts. Our results show that MDH activity could not be detected in our apoplast extract (Table I ). To eliminate the possibility that the difference in MDH activity between the total cell extracts sample and the apoplastic extracts sample was due to the protein concentration difference, the total cell extract was diluted to a concentration that was similar to that of the apoplastic extracts and analyzed again for MDH activity (Table I); the normalized MDH activity in total cell extracts was even higher than in the undiluted samples. In addition, apoplastic extracts from three independent extraction procedures and soluble cell total extracts were separated and probed with the anti-tubulin monoclonal antibody. With an apparent molecular mass of 49 kD, tubulin was detectable in whole cell extracts but not in any of our apoplastic extracts (Fig. 1 ). These results proved that the apoplast extract samples used in this study were free of cytosolic contamination.
The Apoplastic Proteins Changed in Response to Salt Stress Rice root apoplast proteins from the 1-, 3-, and 6-h salt treatments and from the controls were analyzed by 2-DE. Approximately 100 spots from each sample were visualized by Coomassie Brilliant Blue staining (Fig. 2 ). Three independent experiments were conducted to ensure that the protein abundance changes at each time point were reproducible and significant. Software quantification showed that, although the protein expression profiles between the salt-stressed and untreated samples were similar to each other, the abundance of some spots changed significantly with salt stress treatment over time (Figs. 2 and 3 ). We found 10 spots that showed at least a 1.5-fold increase or decrease in abundance (P < 0.05) in response to salt stress (Figs. 2 and 3). Based on their expression profiles, these proteins were classified into three groups. The first group contains spots 1, 2, 5, 6, 7, 8, and 10, which showed a steady increase in abundance over the course of the treatment (Fig. 3). The second group contains spot 9 only, which showed a steady decrease in abundance across all treatment time points (Fig. 3). The third group contains spots 3 and 4. The abundance of the spots in this group increased at 3 h and decreased at 6 h of salt treatment (Fig. 3). The different expression patterns of the spots might imply different roles for these apoplastic proteins in plant salt stress responses.
Identification of Salt Stress-Responsive Proteins by Mass Spectrometry All 10 salt stress-responsive spots were subjected to in-gel digestion and analyzed by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The obtained peptide mass fingerprints were used to search the National Center for Biotechnology Information database using Mascot (http://www.matrixscience.com). The 10 identified proteins spots are listed in Supplemental Table S1. The experimentally calculated molecular mass and pI values of identified protein spots were consistent with the theoretical values, except for spots 1 and 9 (Supplemental Table S1).
To confirm that the proteins we identified were indeed apoplastic proteins, all identified protein sequences were searched using the TargetP program (www.cbs.dtu.dk/services/TargetP) for their predicted subcellular localization (Emanuelsson et al., 2000
Both spots 1 and 6 were identified as protein OsRMC (gi|19387274). To confirm the identities of spots 1 and 6, MS/MS was used for further analysis (Supplemental Figs. S1 and S2). The results showed that spots 1 and 6 represent the same protein, and the pI/molecular mass difference between the two spots may be due to posttranslational modification or dimerization. This protein has been further analyzed using bioinformatics tools. OsRMC belongs to the secreted type of DUF26 protein (Chen, 2001
As OsRMC was obtained from an apoplastic sample and bioinformatics analysis also indicated that OsRMC is a putative secreted protein, we sought experimental evidence for its apoplastic localization. We used Citrine reporter gene, which encodes a new yellow fluorescent protein (YFP) that has lower pH sensitivity (Griesbeck et al., 2001
OsRMC Is a Salt Response Gene and Is Expressed in Different Developmental Stages
The 1,777-bp region upstream of the OsRMC start codon (ATG) was used to search the PLACE database (http://www.dna.affrc.go.jp/PLACE/; Higo et al., 1999
To investigate the transcriptional induction of the OsRMC gene in response to salt stress at the tissue level, transgenic rice lines that expressed the GUS reporter gene under the control of the OsRMC promoter were generated. The promoter was selected from the 1,777-bp sequence upstream of the ATG start codon. A reproducible expression pattern was found in three independent reporter lines. Under standard growth conditions, promoter activity was detected locally in the callus and was high in the roots and leaves of transgenic seedlings (Fig. 5A, a–c). In the mature rice plants, the stems and nodes, except the flag leaf, were stained blue (Fig. 5A, d and e). In the inflorescence, we could see GUS expression in the palea, lodicule, stamen, pistil, and especially in the anther (Fig. 5A, f and g). The seeds of transgenic rice were also stained (Fig. 5A, h and i). Expression of GUS was under the control of the OsRMC promoter in the transgenic plant, so the transcriptional induction of the OsRMC gene in response to salt treatment was determined by monitoring GUS activity. The GUS activity of OsRMCpro::GUS plants in response to salt stress was measured over a 24-h period. Each data point is an average of three independent experiments. Treating the plants with 200 mM NaCl resulted in rapidly and continuously increased GUS activity (Fig. 5C). To further confirm the protein level change of OsRMC in response to salt stress, we generated polyclonal antibodies against OsRMC. A western blot was used to confirm the induction of OsRMC at the translational level by salt stress. The results showed that the level of OsRMC protein increased when plants were treated with 200 mM NaCl for 1 h and kept increasing after 12 h under NaCl treatment (Fig. 5D). These results indicate that OsRMC is a salt stress-responsive gene and that salt stress promotes the expression of OsRMC at the transcriptional and the translational levels.
To assess the function of OsRMC in the salt stress response in vivo, an RNA interference (RNAi) construct (pTCK303) containing part of the coding sequence fragment (428 bp) to knock down OsRMC expression and an overexpression construct (pCAMBIA1300) containing the OsRMC full-length ORF (without the stop codon) fused to a GUS reporter gene driven by a ubiquitin promoter (Ubi-1pro::OsRMC-Gus) were introduced into rice by Agrobacterium tumefaciens EHA105-mediated transformation. We examined the T-DNA insertions of select transgenic lines by Southern blot (Fig. 6A ). Two independent RNAi homozygous lines (Ri1 and Ri2) and an overexpression homozygous line (OXSi) were used for further analysis. Real-time Q-PCR indicated the OsRMC mRNA expression levels in the RNAi lines were significantly knocked down. However, gene silencing of OsRMC also occurred in the overexpression line OXSi, resulting in a low OsRMC expression level, which was similar to that in the RNAi lines (Fig. 6B). To confirm the protein expression level for the RNAi lines, a western-blot assay was performed using an anti-OsRMC antibody. The results of the western blots matched those of the real-time Q-PCR: the protein expression level of OsRMC in the knockdown lines, including the overexpression silenced line, OXSi, was obviously reduced (Fig. 6C).
To analyze the functions of OsRMC under salinity stress, we tested the effect of NaCl on seed germinations of OsRMC knockdown lines. There were no differences in seed germination rates between the transgenic lines and the wild type under normal condition. We tried gradients of NaCl concentrations from 100 to 200 mM to treat seeds for 7 d and found that Ri1 seeds had a higher germination rate than the wild type, and in 175 mM NaCl treatment the difference was most distinct (Fig. 7, A and B ). In 175 mM NaCl treatment, the seeds of Ri1, Ri2, and OXSi were soaked in NaCl solution on a plate containing a filter for 9 d. Seeds were scored for germination from the 4th d, based on whether or not the shoot length exceeded half of the seed length. In statistical analysis, the germination rates of the Ri1, Ri2, and OXSi transgenic lines were higher than that of the wild type in the presence of 175 mM NaCl (Fig. 7C).
We also put the seeds of knockdown lines Ri1 and OXSi in half-strength Murashige and Skoog (1/2MS) medium containing 50 to 175 mM NaCl or without NaCl as a control to germinate and grow into seedlings. Ten-day-old seedlings were measured for shoot length, fresh weight, and dry weight of the shoot. The knockdown transgenic rice seedlings exhibited no difference in growth compared with wild-type rice seedlings under normal conditions, whereas in the presence of NaCl, the knockdown lines showed inhibited growth. In the 100 mM NaCl treatment, the difference was most distinct (Fig. 8A ). Statistical analysis revealed that the knockdown plants exhibited distinct reduction in shoot length, fresh weight, and dry weight compared with control, wild-type plants (Fig. 8, B–D). In 175 mM NaCl, both the germinated seedlings of knockdown plants and control plants exhibited growth reduction with no apparent difference. The Ri2 knockdown line, which has a relatively higher expression level of OsRMC than Ri1 and OXSi (Fig. 6C), was also used for the growth assay in salt treatments and showed a similar phenotype of growth inhibition (data not shown).
Growth rate reduction in the OsMRC-silenced plants could reflect either an adaptive response or an injury response of the plants under the stress (Zhu, 2001 ). In order to delineate these possibilities, we measured the seedling survival rates of OsRMC knockdown lines under salt stress conditions. For the survival assay, 10-d-old knockdown seedlings cultured in Hoagland liquid medium were transferred to Hoagland medium with gradient NaCl concentration from 100 to 200 mM for 4 d and then allowed to recover under normal conditions for 7 d, after which the survival rate was calculated. Plants that could not grow any more after the recovery period were considered to be dead. After recovering for 1 week, more transgenic seedlings survived than wild-type plants, which appeared mostly withered (Fig. 9A
). The survival rates of Ri1 and OXSi were higher than that of the wild type after 7 d of recovery from 150 and 200 mM salt stress, whereas in recovery after 100 mM NaCl treatment both the knockdown lines and the wild type all survived (Fig. 9B). We measured the lipid peroxidation level in rice seedlings under salt stress, and the end product MDA contents of leaves were determined by thiobarbituric acid test (Heath and Parker, 1968). As shown in Figure 9C, MDA levels were elevated in both wild-type and transgenic plants within gradient NaCl treatments, but the degree of lipid peroxidation was significantly lower in knockdown plants than in wild-type plants, which means that the transgenic plants are more tolerant to stress injury.
The expression levels of two salt-responsive genes, OsDREB2A and Rab16A, also were monitored by real-time Q-PCR analysis. As shown in Figure 9D, both OsDREB2A and Rab16A showed higher expression in knockdown lines than in the wild type under normal conditions. When treated with 200 mM NaCl for 6 h, significant inductions of these two salt-responsive genes happened in all salt-treated plants, with higher expression levels in transgenic plants than in wild-type plants. Thus, knockdown of OsRMC results in up-regulated expression of OsDREB2A and Rab16A, which are involved in plant tolerance to salt stress.
To understand the signaling network underlying plant salt adaptation, a two-dimensional electrophoresis approach was used to identify the apoplastic components involved in this process. Despite the difficulties encountered in extracting and identifying proteins, apoplast proteomics has become an active field in recent years (Jamet et al., 2006 , 2008). For a differential display analysis of apoplast proteins responding to salt stress, the quality of the apoplast-specific extract is crucial. There are several methods commonly used to extract the apoplast solution from the plant, including the application of pressure using a Scholander bomb, the vacuum perfusion method, the elution method, and the vacuum infiltration method (Lohaus et al., 2001; Jamet et al., 2008). Of all these methods, the vacuum infiltration method has been most widely used in obtaining apoplastic fluid from different plant species (Husted and Schjoerring, 1995; Lohaus et al., 2001; Dani et al., 2005). To avoid the cytoplasmic contamination of apoplast extracts, an MDH assay and a western-blot assay were used here to evaluate cytoplasmic contamination. MDH activity was detected in total protein extract controls but not in apoplastic extracts (Table I). The immunoblotting against tubulin indicated that tubulin was not present in apoplastic extracts but was present in total protein extracts (Fig. 1). Furthermore, all identified protein sequences were analyzed using the TargetP program (www.cbs.dtu.dk/services/TargetP) to predict their subcellular localization (Emanuelsson et al., 2000), since the sensitivity of mass spectrometry is 100 to 1,000 times greater than that of the biochemical assay (Jamet et al., 2006). As we can see from Supplemental Table S1, eight of 10 spots were predicted to be typical secretory proteins with a signal peptide sequence. These data indicate that our preparations of apoplastic proteins from the rice root did not show detectable intracellular contamination and therefore were appropriate for use in proteomic analysis.
Based on the 2-DE analysis, it was observed that the apoplastic proteins changed in response to salt stress during the initial phase. We found that protein expression pattern changes within 1 h (Figs. 2 and 3), which is quite a swift response. Previous studies had indicated that salt, drought, and heavy metals alter the apoplastic protein composition of leaves (Fuhrer, 1982
Ten significantly salt stress-responsive spots were subjected to in-gel digestion and analyzed by MALDI-TOF MS, and six kinds of protein were identified: OsRMC, a peroxidase, a glucanase, rab5B, a thioredoxin, and a putative pathogenesis-related protein. Based on their potential physiological functions, the salt stress-regulated apoplastic proteins identified in this study are all involved in the plant stress response. In plants, β-1,3-glucanase belongs to the same family of pathogenesis-related proteins as PR-2 and is strongly induced when plants respond to wounding or infection by fungal, bacterial, or viral pathogens (Trudel et al., 1998
It was interesting that spots 1 and 6 were both identified as an OsRMC, containing a predicted N-terminal signal peptide and two DUF26 domains (Supplemental Fig. S3C). Proteins with the DUF26 domain belong to a large protein family with many members in higher plants. All of these share similarities in the DUF26 domain (C-X8-C-X2-C, a Cys-rich repeat motif). These conserved Cys residues may function to maintain the three-dimensional structure and form a zinc finger motif to mediate protein-protein interactions or sense the redox changes in the extracellular space during plant defense responses (Chen, 2001
The sequences of various cis-acting elements, including ABREs, DREs, LTREs, MYBRS, and MYCRS, were predicted in the 1,777-bp promoter region of OsRMC (Supplemental Fig. S3D). These cis elements and their respective transcription factors have important roles in abiotic stress responses (Nakashima and Yamaguchi-Shinozaki, 2006
In the salt stress experiments with transgenic plants, knocking down OsRMC led to growth inhibition upon 50, 100, 150, and 175 mM NaCl treatment for 10 d (Fig. 8, A–D). Although the knockdown lines show higher germination rates than the wild-type control in 175 mM NaCl treatment, in the germinated seedlings there was no apparent difference in growth reduction between the knockdown plants and control plants, both seedlings showing severe growth inhibition in such salt conditions. This result is consistent with the previous work of Jiang et al. (2007)
Under salt stress conditions, the MDA contents in leaves of transgenic rice seedlings were lower than in the wild type, which indicates that the degree of lipid peroxidation was significantly reduced in transgenic plants when treated with salt stress (Fig. 9C). Meanwhile, the rice salt response genes OsDREB2A and Rab16A were up-regulated in the OsRMC knockdown transgenic plants (Fig. 9D). Expression of OsDREB2A was induced by dehydration and high-salt stresses (Dubouzet et al., 2003
Interestingly, it was found that the OsRMC protein was also regulated by pathogen inoculation (Kim et al., 2003
Plant Material and Salt Stress Treatments
Rice (Oryza sativa subsp. japonica ‘Nipponbare’) seedlings were grown in the greenhouse at 28°C/25°C, 16 h of light/8 h of dark, and 50% humidity (Kawasaki et al., 2001
Rice seeds were sterilized and cultured on Murashige and Skoog plus (4 mg L–1 2,4-dichlorophenoxyacetic acid) medium for 4 weeks in the dark at 28°C to induce embryogenic callus for Agrobacterium tumefaciens-mediated transformation, as described by Yang et al. (2004)
Roots were cut into approximately 5-cm segments and washed with deionized water as rapidly as possible. Segments were vacuum infiltrated for 15 min in deionized water, producing a reduced pressure of 80 kPa. The vacuum was gradually released for 5 min. Segments were then dried and carefully arranged in a bundle in a Centriplus concentrator tube (Amicon). The tubes were centrifuged at 900g for 15 min at 4°C to extract the apoplast solution. The apoplast solution was lyophilized and stored at –80°C before analysis. After the apoplastic solution had been extracted, the root tissue was ground to powder and extracted with 50 mM Tris-HCl buffer plus protease inhibitors, stirred for 15 min at 4°C, and centrifuged at 25,000g for 30 min at 4°C. The supernatant was used as total soluble protein for further analysis.
MDH activity was assayed by a modified method from Husted and Schjoerring (1995)
For immunoblots against tubulin, apoplastic proteins and total soluble proteins were separated by 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 or transferred to a polyvinylidene difluoride membrane. The membrane was probed with anti-
After quantification, 250 µL of buffer containing 150 µg of protein was loaded onto 13-cm isoelectric focusing strips, pH 3 to 10 linear gradient (Amersham Biosciences). 2-DE was carried out as described (Blum et al., 1987
The stained gels were scanned using Labscan software with image scanner (Amersham Biosciences). All experiments were repeated at least three times. Then, the gels were analyzed using Image Master 2D Elite software version 4.01 (Amersham Biosciences). After spot detection and normalization (in the total spot volume mode), the protein spots were matched and their volume or abundance was determined. A criterion of P < 0.05 was used to define significant differences when analyzing parallel spots between groups with one-way ANOVA using GraphPad Prim4 (GraphPad Software).
In-gel digestion was performed according to Fulda et al. (2000)
To construct 35S::OsRMC-YFP transient expression vector, the OsRMC 774-bp coding sequence containing a full-length ORF without a stop codon was amplified by reverse transcription (RT)-PCR, digested by NcoI, and ligated to the pAVA321 vector (von Arnim et al., 1998
Histochemical analysis of GUS activity was according to Jefferson et al. (1987)
The plasmids of the OsRMC-Citrine fusion protein construct and Citrine empty vector, which was used as a control, were transformed into the onion (Allium cepa) epidermal cells by particle bombardment in the Bio-Rad PDS-1000/He system according to the protocol. The transformed epidermal cells were cultured on Murashige and Skoog medium for 22 h and then treated with 0.9 M mannitol for plasmolysis. Observation was performed with a confocal microscope (Zeiss 510), and digital images were recorded.
The shoots of 10-d-old rice seedlings were collected for total RNA extraction (Trizol reagent; Invitrogen). Five hundred nanograms of total RNA was used as template for the RT reaction to synthesize the first-strand cDNA, which was used for SYBR Green-monitored real-time Q-PCR as described in the protocol. The analysis was performed with the use of an ABI PRISM 7000 real-time PCR system (Applied Biosystems).
The shoots of 10-d-old rice seedlings were collected for genomic DNA isolation and purification as described by Sambrook et al. (1989)
The OsRMC coding sequence containing a full-length ORF without a stop codon was amplified by RT-PCR, digested by BamHI and EcoRI, and ligated to the pGEX-2T vector. Transformed into Escherichia coli strain BL21, the recombinant proteins extracted from the bacteria were purified on a GSTrap FF column according to the manufacturer's (Amersham Biosciences) protocol. The antibody of OsRMC-GST was prepared by the Tailun Biological Technology company and tested by western blot.
The leaves of 10-d-old rice seedlings were weighed and homogenized in 1 mL of 10% TCA solution. The homogenate was centrifuged, and the supernatant was added to 0.6% thiobarbituric acid in 10% TCA. The mixture was incubated in boiling water for 15 min, and the reaction was stopped in an ice bath. Then, the samples were centrifuged and the absorbance of the supernatant was measured at 450, 532, and 600 nm. MDA contents (nmol g–1 fresh weight) were calculated by the following formula: [6.45(A532 – A600) – 0.56A450]/fresh weight. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AAL87185.
The following materials are available in the online version of this article.
We thank Dr. Kang Chong from the Institute of Botany, Chinese Academy of Sciences, for providing the pTCK303 vector. Received October 11, 2008; accepted November 22, 2008; published November 26, 2008.
1 This work was supported by the Chinese Key National Basic Research and Development Program (grant nos. 2006CB100100 and 2006CB910600), the National Science Foundation of China (grant nos. 30600043 and 30870200), and the Natural Science Foundation of Hebei Province in China (grant nos. C2006000142 and C2008000171). 
2 These authors contributed equally to the article. The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yi Guo (guoyi{at}mail.hebtu.edu.cn).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.131144 * Corresponding author; e-mail guoyi{at}mail.hebtu.edu.cn.
Abbasi FM, Komatsu S (2004) A proteomic approach to analyze salt-responsive proteins in rice leaf sheath. Proteomics 4: 2072–2081[CrossRef][Web of Science][Medline] Acharya BR, Raina S, Maqbool SB, Jagadeeswaran G, Mosher SL, Appel HM, Schultz JC, Klessig DF, Raina R (2007) Overexpression of CRK13 an Arabidopsis cysteine-rich receptor-like kinase results in enhanced resistance to Pseudomonas syringae. Plant J 50: 488–499[CrossRef][Web of Science][Medline] Besse I, Buchanan BB (1997) Thioredoxin-linked plant and animal processes: the new generation. Bot Bull Acad Sin 38: 1–11 Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99[CrossRef][Web of Science] Bolte S, Schiene K, Dietz KJ (2000) Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress. Plant Mol Biol 42: 923–935[CrossRef][Web of Science][Medline] Brune A, Urbach W, Dietz KJ (1994) Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance. Plant Cell Environ 17: 153–162[Medline] Chen K, Du L, Chen Z (2003) Sensitization of defense responses and activation of programmed cell death by a pathogen-induced receptor-like protein kinase in Arabidopsis. Plant Mol Biol 53: 61–74[CrossRef][Web of Science][Medline] Chen K, Fan B, Du L, Chen Z (2004) Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from Arabidopsis. Plant Mol Biol 56: 271–283[CrossRef][Web of Science][Medline] Chen Z (2001) A superfamily of proteins with novel cysteine-rich repeats. Plant Physiol 126: 473–476 Ciftci-Yilmaz S, Morsy MR, Song L, Coutu A, Krizek BA, Lewis MW, Warren D, Cushman J, Connolly EL, Mittler R (2007) The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress. J Biol Chem 282: 9260–9268 Clark SE, Williams RW, Meyerowitz EM (1997) The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89: 575–585[CrossRef][Web of Science][Medline] Dani V, Simon WJ, Duranti M, Croy RRD (2005) Changes in the tobacco leaf apoplast proteome in response to salt stress. Proteomics 5: 737–745[CrossRef][Web of Science][Medline] del Carmen Córdoba-Pedregosa M, Córdoba F, Villalba JM, González-Reyes JA (2003) Zonal changes in ascorbate and hydrogen peroxide contents, peroxidase, and ascorbate-related enzyme activities in onion roots. Plant Physiol 131: 697–706 DeWald DB, Torabinejad J, Jones CA, Shope JC, Cangelosi AR, Thompson JE, Prestwich GD, Hama H (2001) Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed Arabidopsis. Plant Physiol 126: 759–769 Dietz KJ (1997) Functions and responses of the leaf apoplast under stress. Prog Bot 58: 221–254 Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33: 751–763[CrossRef][Web of Science][Medline] Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Mol Biol 300: 1005–1016[CrossRef] Fuhrer J (1982) Ethylene biosynthesis and cadmium toxicity in leaf tissue of beans (Phaseolus vulgaris L.). Plant Physiol 70: 162–167 Fulda S, Huang F, Nilsson F, Hagemann M, Norling B (2000) Proteomics of Synechocystis sp strain PCC 6803: identification of periplasmic proteins in cells grown at low and high salt concentrations. Eur J Biochem 267: 5900–5907[Web of Science][Medline] Gorg A, Boguth G, Obermaier C, Weiss W (1998) Two-dimensional electrophoresis of proteins in an immobilized pH 4 Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein: mechanism and applications. J Biol Chem 276: 29188–29194 Haffani YZ, Silva NF, Goring DR (2004) Receptor kinase signaling in plant. Can J Bot 82: 1–15[CrossRef] Heath RL, Parker L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189–198[CrossRef][Web of Science][Medline] Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27: 297–300 Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM ATAF and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA 35: 12987–12992 Husted S, Schjoerring JK (1995) Apoplastic pH and ammonium concentration in leaves of Brassica napus L. Plant Physiol 109: 1453–1460[Abstract] Jamet E, Albenne C, Boudart G, Irshad M, Canut H, Pont-Lezica R (2008) Recent advances in plant cell wall proteomics. Proteomics 8: 893–908[CrossRef][Web of Science][Medline] Jamet E, Canut H, Boudart G, Pont-Lezica RF (2006) Cell wall proteins: a new insight through proteomics. Trends Plant Sci 11: 33–39[CrossRef][Web of Science][Medline] Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907[Web of Science][Medline] Jiang J, Li J, Xu Y, Han Y, Bai Y, Zhou G, Lou Y, Xu Z, Chong K (2007) RNAi knockdown of Oryza sativa root meander curling led to altered root development and coiling which were mediated by jasmonic acid signalling in rice. Plant Cell Environ 30: 690–699[Medline] Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ (2001) Gene expression profiles during the initial phase of salt stress in rice. Plant Cell 13: 889–905 Kim ST, Cho KS, Yu S, Kim SG, Hong JC, Han CD, Bae DW, Nam MH, Kang KY (2003) Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells. Proteomics 3: 2368–2378[CrossRef][Web of Science][Medline] Koornneef A, Pieterse CMJ (2008) Cross talk in defense signaling. Plant Physiol 146: 839–844 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Lease KA, Walker JC (2006) The Arabidopsis unannotated secreted peptide database, a resource for plant peptidomics. Plant Physiol 142: 831–838 Lee S, Lee EJ, Yang EJ, Lee JE, Park AR, Song WH, Park OK (2004) Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell 16: 1378–1391 Liu J, Ishitani M, Halfter U, Kim CS, Zhu JK (2000) The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci USA 97: 3730–3734 Lohaus G, Pennewiss K, Sattelmacher B, Hussmann M, Muehling KH (2001) Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species. Physiol Plant 111: 457–465[CrossRef][Medline] Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, et al (2007) CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res 35: D237–D240 Matsubayashi Y, Sakagami Y (1996) Phytosulfokine sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 93: 7623–7627 Mikolajczyk M, Awotunde OS, Muszynska G, Klessig DF, Dobrowolska G (2000) Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell 12: 165–178 Misas-Villamil JC, van der Hoorn RAL (2008) Enzyme-inhibitor interactions at the plant-pathogen interface. Curr Opin Plant Biol 11: 1–9[Medline] Mizoguchi T, Ichimura K, Yoshida R, Shinozaki K (2000) MAP kinase cascades in Arabidopsis: their roles in stress and hormone responses. Results Probl Cell Differ 27: 29–38[Medline] Moller S, Croning MDR, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17: 646–653 Mundy J, Chua NH (1988) Abscisic acid and water-stress induce the expression of a novel rice gene. EMBO J 7: 2279–2286[Web of Science][Medline] Munnik T, Ligterink W, Meskiene I, Calderini O, Beyerly J, Musgrave A, Hirt H (1999) Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress. Plant J 20: 381–388[CrossRef][Web of Science][Medline] Munnik T, Meijer HJ, Ter Riet B, Hirt H, Frank W, Bartels D, Musgrave A (2000) Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. Plant J 22: 147–154[CrossRef][Web of Science][Medline] Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51: 617–630[CrossRef][Web of Science][Medline] Nakashima K, Yamaguchi-Shinozaki K (2002) Use of β-glucuronidase to show dehydration and high-salt gene expression. In JF Jackson, HF Linsken, RB Inman, eds, Molecular Methods of Plant Analysis, Vol 22. Testing for Genetic Manipulation in Plants. Springer-Verlag, Berlin, pp 37–61 Nakashima K, Yamaguchi-Shinozaki K (2006) Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants. Physiol Plant 126: 62–71[CrossRef] Ndimba BK, Chivasa S, Hamilton JM, Simon WJ, Slabas AR (2003) Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors. Proteomics 3: 1047–1059[CrossRef][Web of Science][Medline] Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10: 1–6 Nielsen KH, Schjoerring JK (1998) Regulation of apoplastic NH4+ concentration in leaves of oilseed rape. Plant Physiol 118: 1361–1368 Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253: 895–898 Preuss ML, Serna J, Falbel TG, Bednarek SY, Nielsen E (2004) The Arabidopsis Rab GTPase RabA4b localizes to the tips of growing root hair cells. Plant Cell 16: 1589–1603 Qin F, Kakimoto M, Sakuma Y, Maruyama K, Osakabe Y, Tran LS, Shinozaki K, Yamaguchi-Shinozaki K (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. Plant J 50: 54–69[CrossRef][Web of Science][Medline] Ramanjulu S, Kaiser W, Dietz KJ (1999) Salt and drought stress differentially affect the accumulation of extracellular proteins in barley. Z Naturforsch 54: 337–347 RoyChoudhury A, Roy C, Sengupta DN (2007) Transgenic tobacco plants overexpressing the heterologous lea gene Rab16A from rice during high salt and water deficit display enhanced tolerance to salinity stress. Plant Cell Rep 26: 1839–1859[CrossRef][Web of Science][Medline] Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136: 2734–2746 Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18: 1292–1309 Salekdeh GH, Siopongco J, Wade LJ, Ghareyazie B, Bennett J (2002) Proteomic analysis of rice leaves during drought stress and recovery. Proteomics 2: 1131–1145[CrossRef][Web of Science][Medline] Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schopfer CR, Nasrallah ME, Nasrallah JB (1999) The male determinant of self-incompatibility in Brassica. Science 286: 1697–1700 Shen S, Jing Y, Kuang T (2003) Proteomics approach to identify wound-response related proteins from rice leaf sheath. Proteomics 3: 527–535[CrossRef][Web of Science][Medline] Swapan KD, Muthukrishnan S (1999) Pathogenesis Related Proteins in Plants. CRC Press, New York, pp 1–278 Takeda H, Kotake T, Nakagawa N, Sakurai N, Nevins DJ (2003) Expression and function of cell wall-bound cationic peroxidase in asparagus somatic embryogenesis. Plant Physiol 131: 1765–1774 Trudel J, Grenier J, Potvin C, Asselin A (1998) Several thaumatin-like proteins bind to β-1,3-glucans. Plant Physiol 118: 1431–1438 Ueda T, Yamaguchi M, Uchimiya H, Nakano A (2001) Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO J 20: 4730–4741[CrossRef][Web of Science][Medline] Vieira Dos Santos C, Rey P (2006) Plant thioredoxins are key actors in the oxidative stress response. Trends Plant Sci 11: 329–334[CrossRef][Web of Science][Medline] von Arnim AG, Deng XW, Stacey MG (1998) Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221: 35–43[CrossRef][Web of Science][Medline] Wang Z, Chen C, Xu Y, Jiang R, Han Y, Xu Z, Chong K (2004) A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L). Plant Mol Biol Rep 22: 409–417[CrossRef][Web of Science] Xiong L, Yang Y (2003) Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell 15: 745–759 Yan SP, Tang ZC, Sun WA, Sun WN (2005) Proteomic analysis of salt stress-responsive proteins in rice root. Proteomics 5: 235–244[CrossRef][Web of Science][Medline] Yang Y, Peng H, Huang H, Wu J, Jia S, Huang D, Lu T (2004) Large-scale production of enhancer trapping lines for rice functional genomics. Plant Sci 167: 281–288[CrossRef][Web of Science] Young SA, Guo A, Guikema JA, White FF, Leach JE (1995) Rice cationic peroxidase accumulates in xylem vessels during incompatible interaction with Xanthomonas oryzae pv. oryzae. Plant Physiol 107: 1333–1341[Abstract] Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6: 66–71[CrossRef][Web of Science][Medline] Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247–273[CrossRef][Medline] This article has been cited by other articles:
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ABSTRACT
RESULTS
-tubulin monoclonal antibody. Lane 1 contains a protein molecular mass standard.
12 gradient. Electrophoresis 19: 1516–1519
