Soil salinity is a growing problem around the world with special relevance in farmlands. The ability to sense and respond to environmental stimuli is among the most fundamental processes that enable plants to survive. At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway that comprises SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress, to maintain ion homeostasis. Less well known is how cellularly heterogenous organs couple the salt signals to homeostasis maintenance of different types of cells and to appropriate growth of the entire organ and plant. Recent evidence strongly indicates that different regulatory mechanisms are adopted by roots and shoots in response to salt stress. Several reports have stated that, in roots, the SOS proteins may have novel roles in addition to their functions in sodium homeostasis. SOS3 plays a critical role in plastic development of lateral roots through modulation of auxin gradients and maxima in roots under mild salt conditions. The SOS proteins also play a role in the dynamics of cytoskeleton under stress. These results imply a high complexity of the regulatory networks involved in plant response to salinity. This review focuses on the emerging complexity of the SOS signaling and SOS protein functions, and highlights recent understanding on how the SOS proteins contribute to different responses to salt stress besides ion homeostasis.

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

The phytohormone abscisic acid (ABA) plays a central role in plant development and in plant adaptation to both biotic and abiotic stressors. In recent years, knowledge of ABA metabolism and signal transduction has advanced rapidly to provide detailed glimpses of the hormone's activities at the molecular level. Despite this progress, many gaps in understanding have remained, particularly at the early stages of ABA perception by the plant cell. The search for an ABA receptor protein has produced multiple candidates, including GCR2, GTG1, and GTG2, and CHLH. In addition to these candidates, in 2009 several research groups converged on a novel family of Arabidopsis proteins that bind ABA, and thereby interact directly with a class of protein phosphatases that are well known as critical players in ABA signal transduction. The PYR/PYL/RCAR receptor family is homologous to the Bet v 1-fold and START domain proteins. It consists of 14 members, nearly all of which appear capable of participating in an ABA receptor–signal complex that responds to the hormone by activating the transcription of ABA-responsive genes. Evidence is provided here that PYR/PYL/RCAR receptors can also drive the phosphorylation of the slow anion channel SLAC1 to provide a fast and timely response to the ABA signal. Crystallographic studies have vividly shown the mechanics of ABA binding to PYR/PYL/RCAR receptors, presenting a model that bears some resemblance to the binding of gibberellins to GID1 receptors. Since this ABA receptor family is highly conserved in crop species, its discovery is likely to usher a new wave of progress in the elucidation and manipulation of plant stress responses in agricultural settings.

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 salt-stressed 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.

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 H₂O₂ 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 H₂O₂ are involved in salt resistance. However, the double mutant did not hyperaccumulate H₂O₂ in response to salt stress, suggesting that it is altered signaling rather than H₂O₂ 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 H₂O₂ 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 H₂O₂.

Anthocyanins are major pigments in colored grape (Vitis vinifera) berries, and most of them are monomethoxylated or dimethoxylated. We report here the functional characterization of an anthocyanin O-methyltransferase (AOMT) from grapevine. The expression pattern in two cultivars with different anthocyanin methylation profiles (Syrah and Nebbiolo) showed a peak at start ripening (véraison), when the concentrations of all methylated anthocyanins begin to increase. The purified recombinant AOMT protein was active on both anthocyanins and flavonols in vitro, with K m in the micromolar range, and was dependent on divalent cations for activity. AOMT showed a preference for 3′,5′ methylation when a 3′,4′,5′ hydroxylated anthocyanin substrate was tested. In order to assess its in planta activity, we performed transient expression of AOMT in tobacco (Nicotiana benthamiana) leaves expressing the Production of Anthocyanin Pigment1 (PAP1) transcription factor from Arabidopsis (Arabidopsis thaliana). PAP1 expression in leaves induced the accumulation of the nonmethylated anthocyanin delphinidin 3-rutinoside. The coexpression of PAP1 and AOMT resulted in an accumulation of malvidin 3-rutinoside. We also showed that AOMT localized exclusively in the cytoplasm of tobacco leaf cells. These results demonstrate the ability of this enzyme to methylate anthocyanins both in vitro and in vivo, indicating that AOMT plays a major role in anthocyanin biosynthesis in grape berries.