The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H ؉ -ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and saltstressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H ؉ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.To cope with salt stress, plants have evolved strategies to maintain low Na ϩ concentrations in the cytoplasm. The salt overly sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress (63, 64). SOS1 is a Na ϩ /H ϩ exchanger located on the plasma membrane (39, 53); SOS3 is a myristoylated EF hand-type Ca 2ϩ -binding protein able to sense specific salt stress-induced calcium signals (19), and SOS2 is a Ser/Thr kinase with a C-terminal regulatory domain and an N-terminal catalytic domain (24). During salt stress conditions, the SOS2-SOS3 complex phosphorylates and activates the transport activity of the SOS1 antiporter (42).The function of the SOS2-SOS3 regulatory complex depends on interaction of SOS2 and regulatory proteins, including SOS3. The C-terminal regulatory domain of SOS2 consists of an autoinhibitory FISL motif that binds to SOS3 (13, 24) and a PPI motif that binds to type 2C protein phosphatase abcisic acid (ABA)-insensitive 2 (ABI2) (33). Yeast two-hybrid experiments have shown that the SOS2 protein physically interacts with SOS3, and in vitro phosphorylation assays have shown that Ca 2ϩ is required to activate the kinase activity of the SOS2-SOS3 complex (16). SOS3 binding also recr...

SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H 2 O 2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H 2 O 2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H 2 O 2 in response to salt stress, suggesting that it is altered signaling rather than H 2 O 2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H 2 O 2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H 2 O 2 .Among the mechanisms known to be important in abiotic stress responses in plants are the salt overly sensitive (SOS) ion homeostasis and signaling pathway (66, 67) and reactive oxygen species (ROS) accumulation and signaling (16,41). The SOS pathway is currently one of the most extensively studied mechanisms in controlling salt stress response in plants. The SOS1, SOS2, and SOS3 loci were first identified through forward genetic screens for salt-hypersensitive growth (67). SOS1 is a plasma membrane Na ϩ /H ϩ antiporter that is essential for Na ϩ efflux from roots (48, 55). SOS2 belongs to subgroup 3 of the sucrose-nonfermentingrelated kinases (SnRK3s) (34), of which there are 25 encoded by the Arabidopsis thaliana genome (27). SOS3 is a myristoylated calcium-binding protein that likely responds to salt-induced Ca 2ϩ oscillations in the cytosol (33). A total of nine SOS3-like Ca 2ϩ binding proteins (SCaBPs) are encoded by the Arabidopsis genome (21).The SOS2 kinase has emerged as an especially important regulatory component through its interactions with other signaling proteins. First, as part of the SOS signaling pathway, the regulatory region of SOS2 was shown to interact with SOS3 (25). This interaction activates SOS2 protein kinase activity in a Ca 2ϩ -dependent manner and recruits the SOS2-SOS3 complex to the plasma membrane, where it phosphorylates SOS1 and activates Na ϩ efflux (48,50). Specific interactions between other SnRK3s (also referred to as calcineurin B-like proteininteracting protein kinases [CIPK]) and SCaBPs (also referred to as calcineurin B-like proteins [CBL]) have also been detected and are involved in signal transduction controlling abcisic acid (ABA) sensitivity, cold response, sugar response, and cellular pH (6,11,21,32,46). Previous work has also shown that SOS2 interacts with the ABA-insensitive 2 (ABI...

The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2-to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.