Soil salinity is one of the most significant abiotic stresses, especially for crop plants, leading to reductions in productivity. Salt stress causes accumulation of excess toxic Na⁺, along with deficiency of K⁺, and turgor changes in the cytosol, which in turn induce ionic and osmotic stress in plants, respectively. Salt-induced ionic stress is clearly distinct from other types of stress, whereas osmotic stress is generally induced by salt, cold, and drought. Plant cells have the capacity to adapt to stress conditions by triggering a network of signaling events.

Genetic analyses have led to the elucidation of the salt overly sensitive (SOS) signaling pathway that controls ionic stress responses (Wu et al., 1996; Liu and Zhu, 1997; Zhu et al., 1998). SOS3, a Ca²⁺ binding protein, senses the Ca²⁺ change elicited by salt stress (Quintero et al., 2002). The protein physically interacts with and activates SOS2, a Ser/Thr protein kinase, in a Ca²⁺-dependent manner (Halfter et al., 2000). The SOS3-SOS2 kinase complex regulates the expression and transport activity of ion transporters such as SOS1, a plasma membrane Na⁺/H⁺ exchanger, eventually removing Na⁺ from the cytosol (Qiu et al., 2002).

Evidence has been presented showing that the osmotic stress response is mediated by signaling pathways distinct from the SOS pathway, with the identification of several protein kinases activated by osmotic stress (Zhu, 2002). Mitogen-activated protein kinases (MAPKs) are activated by hyperosmotic stress (Xiong et al., 2002). Specific MAPKs, such as salt stress–inducible MAPK and salicylic acid–induced protein kinase, are present in alfalfa (Medicago sativa) and tobacco (Nicotiana tabacum) cells, respectively (Munnik et al., 1999; Mikolajczyk et al., 2000). In Arabidopsis thaliana, at least three MAPKs are activated by salt and other stresses (Ichimura et al., 2000; Droillard et al., 2002). Ca²⁺-dependent protein kinases have also been implicated in the osmotic stress response in association with Ca²⁺ signaling (Romeis et al., 2001). The plant hormone abscisic acid (ABA) has long been known to play a critical role in stress responses (Giraudat et al., 1994; Himmelbach et al., 2003). Whereas osmotic and cold stresses induce increased levels of ABA (Zeevaart and Creelman, 1998), some osmotic stress–responsive genes are induced by ABA (Skriver and Mundy, 1990). In addition, phospholipid signaling is closely related to osmotic stress (Zhu, 2002). Osmotic stress, cold, and ABA activate phospholipases that generate the second messengers, inositol 1,4,5-trisphosphate, diacylglycerol, and phosphatidic acid, which act in signaling pathways implicated in stress resistance (Dove et al., 1997; Munnik et al., 2000; DeWald et al., 2001). ABA and phospholipid molecules appear to function upstream of the osmotic stress–activated protein kinases. In plants, different signaling processes are integrated to cope with osmotic stress.

ABA, cold, drought, and salt stress trigger elevations in the cytosolic Ca²⁺ level in plant cells (Knight et al., 1996; Knight and Knight, 2001). As a second messenger, Ca²⁺ activates signaling pathways and therefore influences multiple aspects of cellular functions (Knight et al., 1996, 1997; Trewavas, 1999). Ca²⁺ binding proteins serve as transducers of the Ca²⁺ signal. Ca²⁺ binding proteins have been identified in plants, such as calmodulin (Zielinski, 1998; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harmon et al., 2000; Romeis et al., 2001), calcineurin B–like proteins (Luan et al., 2002), and SOS3 (Liu and Zhu, 1998). Certain of these proteins are involved in ABA and abiotic stress responses (Sheen, 1996; Sajio et al., 2000; Townely and Knight, 2002).

Despite considerable progress in understanding stress signal transduction, the mechanisms of stress response remain largely unknown (Xiong et al., 2002). The identification of novel signaling components should contribute to the clarification of stress signaling. After the completion of genome sequencing in Arabidopsis, the identification of stress-responsive proteins is currently feasible with proteomics. In this study, the microsomal proteome from Arabidopsis roots was isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). In an attempt to identify the membrane proteins involved in salt stress, we evaluated salt-induced changes in the microsomal proteome and identified Ca²⁺-dependent membrane binding proteins, designated annexins, as the signaling components of the stress response. Two-dimensional gel analyses combined with protein gel blotting revealed that levels of annexin 1 (AnnAt1) significantly increase in the microsome in a Ca²⁺-dependent manner in response to osmotic stress. The annAt1 and annAt4 mutant plants were hypersensitive to salt and ABA during seed germination and early seedling growth. Based on these findings, we propose that annexins comprise a novel class of Ca²⁺ binding proteins that play important roles in ABA-mediated stress response in plants.

A 2D gel of root microsomal proteins revealed ~350 protein spots evenly distributed between pH 4 and 7 and molecular masses of 10 to 120 kD (Figure 1A). We randomly selected and identified spots with MALDI-TOF MS (Figure 1A, Table 1). The most prominent proteins were identified as mitochondrial and vacuolar ATPases. To analyze the salt response of root microsomal proteins, changes in spot intensity between untreated and treated samples were quantified by software analysis (see Methods). Protein spot changes were scored only when they were reproducibly observed in three independent experiments. Of the protein spots displaying greater than twofold upregulation or downregulation, six (spot numbers 21, 33, 34, 38, 96, and 97) were subjected to identification with MALDI-TOF MS analysis (Figure 1B, Table 1).

Figure 1. Two-Dimensional Gel Electrophoresis Analysis of Root Microsomal Proteins.

Table 1. Identification of Root Microsomal Proteins in Arabidopsis Using MALDI-TOF MS

Among the salt-responsive proteins, p33 and p34 (spot numbers 33 and 34) representing AnnAt1 were initially selected for further characterization. AnnAt1 is an interesting molecule for several reasons. First, annexins participate in essential cellular processes in animal cells (Gerke and Moss, 2002). Second, their properties are directly regulated by Ca²⁺ that is implicated in stress response in plants (Knight et al., 1996; Knight and Knight, 2001). p33 and p34 protein spots migrated with a molecular mass of 40 kD, which is slightly larger than the theoretical molecular size of AnnAt1 (36 kD). The apparent pI values of p33 and p34 on a 2D gel are 5.2 and 5.3, consistent with the theoretical pI (5.2).

Figure 2. Expression of AnnAt1 in Tissues.

Figure 3. Expression of AnnAt1 in Response to Abiotic Stress.

The expression pattern of AnnAt1 in tissues was determined by protein gel blot analysis. AnnAt1 was expressed predominantly in root tissue (Figure 2). The immunodetectable level of AnnAt1 in roots from Arabidopsis grown in soil was similar to that in Arabidopsis roots cultured in MS media used throughout the experiments.

Next, we examined whether AnnAt1 expression is affected by ABA and other stress. We found that AnnAt1 protein was induced by ABA (Figure 3C). Treatment with mannitol and polyethylene glycol (PEG) additionally elevated AnnAt1 levels (Figure 3C), suggesting that the protein is sensitive to ABA and general osmotic stress.

Immunoblotting of 2D gels with the anti-AnnAt1 antibody revealed at least four spots, including p33 and p34 (Figures 3 and 4). Two additional 40-kD spots are unlikely to be other members of the annexin gene family that have the theoretical pI values between 5.8 and 9.5. To verify that the additional spots represent AnnAt1 protein, the spots were eluted from the gel and subjected to MALDI-TOF MS. Peptide masses from the spectra matched that of AnnAt1 in the database search, indicating that all four spots represent AnnAt1 protein (data not shown).

Figure 4. Salt and Calcium Response of AnnAt1 Protein.

We compared the salt-induced changes in AnnAt1 protein of the microsomal and cytosolic fractions by protein gel blot analysis of 2D gels. Immunodetectable AnnAt1 protein levels were considerably enhanced in the microsome after 2 h of salt treatment and fully recovered at 24 h (Figures 4A and 4E). Notably, the expression pattern was reversed in the cytosol, being almost completely abolished at 2 h of salt treatment and recovered thereafter. The pattern of expression of total AnnAt1 was similar to that of cytosolic AnnAt1, consistent with the fact that the cytosol constitutes the major fraction (>99%) and microsomes are very diluted (<1%) in the total fraction. The microsomal fraction was highly concentrated from the total fraction by ultracentrifugation. The results suggest that salt treatment affects AnnAt1 protein in two ways: specifically, translocation from the cytosol to the membrane and protein turnover in the cytosol.

The Ca²⁺ dependency of the subcellular distribution of AnnAt1 protein was examined. Plant extracts were incubated with either Ca²⁺ or EGTA before fractionation. Ca²⁺ increased the relative amount of AnnAt1 protein associated with the microsomal fraction, whereas EGTA had the opposite effect (Figure 4B). We further investigated the Ca²⁺ effect on salt response of AnnAt1 protein in vivo. The subcellular distribution of AnnAt1 protein was determined in Ca²⁺-depleted plants incubated in MS media containing EGTA. Association with the membrane and Na⁺-stimulated reduction in amount of AnnAt1 protein were both inhibited by EGTA (Figure 4C). AnnAt1 levels were partially affected, possibly because of incomplete Ca²⁺ chelation in plants. The results strongly suggest that the salt response of AnnAt1 protein is mediated by Ca²⁺.

We investigated whether the salt response of AnnAt1 protein is observed at the transcript level. The 3′-untranslated region (UTR) of AnnAt1 that is specific to AnnAt1 in RNA gel blot experiments (Clark et al., 2001) was used as a probe. RNA gel blot analysis revealed that in contrast with the salt-induced changes in protein, the transcript was not affected (Figure 4D). The AnnAt1 level even slightly decreased over time. The data suggest that AnnAt1 is regulated translationally (i.e., by the rate of protein synthesis) or posttranslationally (i.e., by the translocation and turnover of protein).

Figure 5. T-DNA Insertion Mutants of AnnAt Genes.

To determine the exact positions of T-DNA insertions, genomic DNA fragments of AnnAt and T-DNA junctions were amplified and sequenced for annAt1, annAt2, and annAt4 mutants (Figure 5A). The insertions were detected at positions of nucleotides 1192, 1605, 1670, and 1641 in annAt1, annAt2-1, annAt4-1, and annAt4-2, respectively. Furthermore, DNA gel blot analyses revealed that a single insertion is present in these lines (data not shown). In the phenotypic analyses, two independent annAt4 alleles, annAt4-1 and annAt4-2, displayed similar mutant phenotypes (Figures 6D, 7, and 8B), suggesting that a T-DNA insertion into the AnnAt4 gene is responsible for the observed phenotypes. For annAt1, a genetic complementation test was additionally performed. Transformation of the mutant plant with the vector containing AnnAt1 cDNA under the control of the 35S promoter of Cauliflower mosaic virus rescued the phenotypes (Figures 6F, 7E, and 8E). These plants (annAt1/ANNAt1) contained similar levels of AnnAt1, compared with the wild-type plants under normal growth conditions (Figure 5C). The results collectively demonstrate that T-DNA insertions provided knockout alleles of AnnAt1 and AnnAt4 genes.

Figure 6. Sensitivity of annAt Mutant Plants to NaCl.

Figure 7. Sensitivity of annAt1 and annAt4 Mutant Plants to Osmotic Stress.

Figure 8. Sensitivity of annAt1 and annAt4 Mutant Plants to ABA.

Although both annAt1 and annAt4 displayed hypersensitivity to NaCl, slightly different patterns were obtained in response to salt. Unlike annAt1, annAt4-1 and annAt4-2 displayed a sudden decrease in germination at 75 mM NaCl (Figure 6D). As shown in Figure 6E, seed germination of annAt1 and annAt4-1 was significantly affected (80% inhibition) in MS media containing 75 mM NaCl. The growth of germinated annAt1 and annAt4-1 plants was arrested after radicles emerged and resumed upon transfer to MS media. The results collectively suggest that AnnAt1 and AnnAt4 are implicated in salt stress response in plants.

Germination of annAt1 and annAt4-1 plants is inhibited in media containing ABA compared with the wild type and abi1, the ABA-insensitive mutant line (Koornneef et al., 1984) (Figure 8C). Moreover, growth of annAt1 and annAt4-1 plants was impaired after radicles emerged, whereas wild-type plants continued to grow and get green (Figure 8D). We also found that annAt1 and annAt4-1 were in a state of growth arrest in the presence of ABA and resumed normal growth upon transfer to ABA-deficient MS media, as observed with NaCl.

Although AnnAt1 is associated with microsomes, the protein is more abundant in the cytosol. The microsomal fraction comprises membranes originating from different organelles, such as vacuole, chloroplast, Golgi, and plasma membrane. Previous reports indicate that annexins are subcellularly localized in the plasma membrane, vacuole, and nuclear periphery (Clark and Roux, 1995). To confirm subcellular localization, green fluorescent protein–fused AnnAt1 was transiently expressed in BY-2 protoplasts. AnnAt1 was detected in both the cytosol and plasma membrane, and the green fluorescent protein signal was enhanced in the plasma membrane upon salt and ABA treatment (data not shown).

In many signaling processes, regulatory proteins are recruited from the cytosol to the membrane (Didichenko et al., 1996; Park et al., 2000; Oancea et al., 2003). Membrane association is often triggered by posttranslational modifications, such as phosphorylation, lipidation and glycosylation, and/or protein–protein interactions (Iwata et al., 1998). AnnAt1 was observed as at least four spots with different pI values on a 2D gel, suggesting posttranslational modifications. We are currently investigating the possibility of phosphorylation and other modifications of AnnAt1 protein, as evidenced in animal cells (Gerke and Moss, 2002). However, after stress treatment, AnnAt1 spots moved to the membrane together, implying that the possible modifications are not directly related to membrane association and play no functional roles. AnnAt1 spots remaining in the cytosol were indistinguishable from those in the membrane, supporting this finding. The formation of the cluster spots on a 2D gel may be simply because of unknown technical reasons. It is possible that the Ca²⁺-dependent association of AnnAt1 with the membrane could also involve protein–protein interactions. Our preliminary data show that the sizes of the AnnAt1-associated complexes on a native gel differ depending on whether the complexes are isolated from the cytosolic or membrane fraction and on whether the plants are exposed to stress stimuli (data not shown). Therefore, identification of the interacting components in AnnAt1 complexes should facilitate elucidation of the specific functions of the protein and the functional significance of membrane association in stress responses. With regard to protein turnover, proteolysis, particularly the ubiquitin/26S proteasome pathway, is one of the most important regulatory mechanisms controlling cellular functions in plants (Vierstra, 2003). Several known signaling components, including phyA, HY5/HYH, AUX/IAA, NAC1, E2F, and ABI5, have been identified as target substrates. A previous report shows that annexins may be regulated by proteolysis, possibly through the lysosomal pathway, in rat lung tissue (Barnes and Gomes, 2002). Whether AnnAt1 is a selective target for the ubiquitin/26S proteasome or other proteolytic pathways would be an intriguing question.

Annexins are characterized by their ability to bind phospholipids in a Ca²⁺-dependent manner. In this study, we provide evidence that Ca²⁺ mediates the association of AnnAt1 protein with the membrane. The inclusion of Ca²⁺ in plant extracts induced binding of AnnAt1 to the membrane, which was reversed by the addition of EGTA. In plants incubated in Ca²⁺-chelated media, AnnAt1 lost the ability to respond to salt stress because both accumulation in the membrane and loss in the cytosol were inhibited. However, the inhibition of AnnAt1 loss from the cytoplasmic fraction was only partial. We suspect that EGTA in the media was not fully effective in chelating the intracellular Ca²⁺ that is instantly released from Ca²⁺ stores in response to stress (DeWald et al., 2001). These results imply that the salt stress–induced response of AnnAt1 is specifically regulated by Ca²⁺. Alternatively, we cannot rule out the possibility that Ca²⁺ binding may cause conformational changes in AnnAt1 and changes in the solubility or association of this protein with other cytosolic proteins and subsequent aggregation. In addition, several fundamental questions are yet to be solved: specifically, whether the cytosolic and membrane-bound forms of AnnAt1 are structurally and functionally different and the mechanism by which Ca²⁺ induces AnnAt1 loss from the cytoplasmic fraction.

In this study, we demonstrate that AnnAt1 is possibly involved in the osmotic stress response. The annAt1 mutant showed hypersensitivity to ABA and osmotic stress induced by such agents as NaCl, LiCl, CsCl, KCl, and mannitol in germination. In addition to annAt1, annAt4 was defective in germination under stress conditions. Whereas annAt1 and annAt4 plants responded similarly to stress, they exhibited slightly different responses to various osmotic stress, with distinct ion selectivities and kinetics of germination. This may be because of differences in temporal and spatial expression and expression levels of proteins. AnnAt1 and AnnAt4 may have distinct, further defined roles in stress response in plants. However, we failed to detect additional altered phenotypes of annAt1 and annAt4 in response to ABA and osmotic stress, such as root growth inhibition and leaf wilting with growth. The data suggest that AnnAt1 and AnnAt4 may function within a restricted developmental window that includes the germination and early seedling stage. Alternatively, it is possible that the proteins exhibit functional redundancy, substituting for each other in response to stress. In contrast with annAt1 and annAt4, the annAt2 mutant exhibited normal phenotypes similar to those of the wild type in the presence of ABA and osmotic stress. Although Arabidopsis annexins are structurally conserved, their functions may be diverse and regulated in distinct ways.

At present, the mechanism by which AnnAt1 functions in ABA and osmotic stress signaling processes is unclear. It is tempting to speculate that AnnAt1 senses the Ca²⁺ signal elicited by ABA and stress and transmits it to downstream signaling pathways via dual mechanisms of protein degradation and translocation to the membrane. Degradation may release the interacting molecules, and translocation may enhance association with other molecules in the membrane, both resulting in the activation of the downstream signaling cascade. Receptors, channels, and kinases are good candidates for interacting partners. Additional studies will be required to elucidate the biological functions and action mechanisms of plant annexins.

The pellet was dried and dissolved in isoelectric focusing sample buffer (7 M urea, 2 M thiourea, 0.05% dodecylmaltoside, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, 20 mM Tris, 20 mM DTT, 0.5% IPG buffer, and 0.001% bromophenol blue). Immobiline DryStrips (pH 4 to 7, linear; 70 mm for protein gel blotting and 180 mm for MALDI-TOF MS analysis) (Amersham Biosciences, Uppsala, Sweden) were rehydrated with proteins and focused on the IPGphor system (Amersham Biosciences). Strips were transferred to equilibration buffer (50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 20 mM tributylphosphine [TBP]) and incubated for 15 min. When TBP is used as the reducing agent, a single step equilibration takes place using TBP and acrylamide, and iodoacetamide is not needed. The advantage of alkylating with acrylamide is to form a single alkylated product. Equilibrated strips were placed on top of vertical polyacrylaminde gels and overlaid with 0.5% agarose in SDS running buffer. After electrophoresis, 2D gels were stained with silver nitrate according to the manufacturer's manual (Amersham Biosciences). Molecular weight (MBI Fermentas, Vilnius, Lithuania) and pI (Amersham Biosciences) markers were used to calculate apparent molecular masses and pI values of spots. Two-dimensional gels were scanned and analyzed by ImageMaster 2D Elite software (Amersham Biosciences). For each condition analyzed, three to five gels were prepared from three different protein extractions. The volumes of silver-stained spots were normalized to the volumes of internal standards (e.g., spot numbers 20 and 24). The salt-induced change was subjected to statistical analysis with Student's t test, and those spots with P < 0.05 were considered for identification by MALDI-TOF MS.

RT-PCR was performed with 0.4 and 0.1 μg of total RNA for the detection of AnnAt4 and Actin, respectively, using the Access RT-PCR system (Promega, Madison, WI). The primers used to amplify the cDNA fragments were as follows: AnnAt4, 5′-ACACTGGGGAAATCGCAAAAG-3′ and 5′-AGCCAAAGTCTCACCATAAAG-3′; Actin, 5′-GGCGATGAAGCTCAATCCAAACG-3′ and 5′-GGTCACGACCAGCAAGATCAAGACG-3′. The primers produced 801-bp and 491-bp products for AnnAt4 and Actin, respectively.

Sequence data from this article have been deposited with the GenBank/EMBL data libraries under the following accession numbers: AnnAt1 (At1g35720), AnnAt2 (At5g65020), and AnnAt4 (At2g38750).

We are grateful to Moon Soo Soh for technical comments. We also thank Mi-Ok Han for technical assistance. This research was supported by grants from the Crop Functional Genomics Center of 21st Century Frontier Research Program funded by the Ministry of Science and Technology (M101KG010001-03K0701-01620) and the Plant Signaling Network Research Center funded by the Korea Science and Engineering Foundation. This article is Kumho Life and Environmental Science Laboratory publication number 66.