Differential expression of poplar <Emphasis Type="Italic">sucrose nonfermenting1-related protein kinase 2</Emphasis> genes in response to abiotic stress and abscisic acid

Knowledge on the responses of woody plants to abiotic stress can inform strategies to breed improved tree varieties and to manage tree species for environmental conservation and the production of lignocellulosic biomass. In this study, we examined the expression patterns of poplar (Populus trichocarpa) genes encoding members of the sucrose nonfermenting1-related protein kinase 2 (SnRK2) family, which are core components of the abiotic stress response. The P. trichocarpa genome contains twelve SnRK2 genes (PtSnRK2.1- PtSnRK2.12) that can be divided into three subclasses (I–III) based on the structures of their encoded kinase domains. We found that PtSnRK2s are differentially expressed in various organs. In MS medium-grown plants, all of the PtSnRK2 genes were significantly upregulated in response to abscisic acid (ABA) treatment, whereas osmotic and salt stress treatments induced only some (four and seven, respectively) of the PtSnRK2 genes. By contrast, soil-grown plants showed increased expression of most PtSnRK2 genes under drought and salt treatments, but not under ABA treatment. In soil-grown plants, drought stress induced SnRK2 subclass II genes in all tested organs (leaves, stems, and roots), whereas subclass III genes tended to be upregulated in leaves only. These results suggest that the PtSnRK2 genes are involved in abiotic stress responses, are at least partially activated by ABA, and show organ-specific responses.     

Genome-wide identification,characterization, and expression analysis of <Emphasis Type="Italic">SnRK2</Emphasis> family in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family belongs to a group of plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and biotic and abiotic stress response. Although genome-wide analyses of the SnRK2 gene family have been conducted in some species, little is known about the SnRK2 gene family in rubber tree (Hevea brasiliensis). In this study, we identified 10 SnRK2s designated as HbSnRK2.1 to HbSnRK2.10 in the rubber tree genome. The subsequently constructed phylogenetic tree demonstrated that HbSnRK2s have three subfamilies that correlate well with those of Arabidopsis sp. and rice subfamilies. All SnRK2 genes contained nine exons and eight introns. Although the C-terminus was divergent, eight conserved motifs were found. Motifs 1–6 were common to all HbSnRK2s. Expression analysis results showed that 7 of the 10 HbSnRK2s were highly expressed in latex. HbSnRK2.7 was predominantly expressed and simultaneously regulated by abscisic acid, jasmonic acid, and ethylene treatment in laticifers. HbSnRK identification and characterization provided further understanding on the role of ABA signal in the rubber tree.     

Cloning and characterization of the SnRK2 gene family from <Emphasis Type="Italic">Zea mays</Emphasis>

The SnRK2 gene family is a group of plant-specific protein kinases that has been implicated in ABA and abiotic stress signaling. We found 11 SnRK2s in maize, assigned names from ZmSnRK2.1 to ZmSnRK2.11 and cloned ten of them. By analyzing the gene structure of all the SnRK2s from Arabidopsis, rice, and maize, we found seven exons that were conserved in length among most of the SnRK2s. Although the C-terminus was divergent, we found seven conserved motifs. Of these, motif 1 was common to all of the SnRK2 genes. Based on phylogenetic analysis using the kinase domain and motif 1, the SnRK2s were divided into three groups. Motifs 4 and 5 were found specifically in group I, and many genes of this group have been confirmed to be induced by ABA. This result suggests that these two motifs mediate the ABA response. The expression patterns of ZmSnRK2 genes were characterized by using quantitative real-time RCR, which revealed that ZmSnRK2 genes were induced by one or more abiotic stress treatments and therefore may play important roles in maize stress responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Huai and M. Wang have contributed equally to this paper.     

Cloning and characterization of four <Emphasis Type="Italic">TpSnRK2s</Emphasis> from dwarf Polish wheat

Protein phosphorylation/dephosphorylation is a major signalling event induced by abiotic stresses in plants. Sucrose nonfermenting 1-related protein kinase 2 (SnRK2) plays important roles in response to osmotic stress. In the present study, four SnRK2s, TpSnRK2.1/3/7/8, were cloned and characterized from Triticum polonicum L. (dwarf Polish wheat, DPW, AABB). All of these were individually located on 2AL, 1AL, 2AL, and 5BL. Two spliced isoforms of TpSnRK2.8 (TpSnRK2.8a and TpSnRK2.8b) were observed. TpSnRK2.1 and TpSnRK2.3 were classified into the group II; TpSnRK2.7 was classified into the group I; and TpSnRK2.8a/b were classified into the group III. Expression patterns revealed that TpSnRK2.1 responded to cold, NaCl, polyethylene glycol (PEG), and abscisic acid (ABA) in both roots and leaves; TpSnRK2.3 was strongly regulated by cold, NaCl, and ABA in both roots and leaves, and by PEG in roots; TpSnRK2.7 was induced by NaCl and PEG in roots, but was not activated by ABA; and TpSnRK2.8s were significantly activated by cold, NaCl, PEG, and ABA in both roots and leaves. From the above results, we inferred that TpSnRK2.1/3/8 may participate in the responses to environmental stresses in ABA-dependent signal transduction pathway but TpSnRK2.7 is possibly involved in responses to environmental stresses in a non-ABA-dependent manner. They play important roles in specific tissues under different stresses.     

Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) plays a key role in the plant stress signalling transduction pathway via phosphorylation. Here, a SnRK2 member of common wheat, TaSnRK2.7, was cloned and characterized. Southern blot analysis suggested that the common wheat genome contains three copies of TaSnRK2.7. Subcellular localization showed the presence of TaSnRK2.7 in the cell membrane, cytoplasm, and nucleus. Expression patterns revealed that TaSnRK2.7 is expressed strongly in roots, and responds to polyethylene glycol, NaCl, and cold stress, but not to abscisic acid (ABA) application, suggesting that TaSnRK2.7 might participate in non-ABA-dependent signal transduction pathways. TaSnRK2.7 was transferred to Arabidopsis under the control of the CaMV-35S promoter. Function analysis showed that TaSnRK2.7 is involved in carbohydrate metabolism, decreasing osmotic potential, enhancing photosystem II activity, and promoting root growth. Its overexpression results in enhanced tolerance to multi-abiotic stress. Therefore, TaSnRK2.7 is a multifunctional regulatory factor in plants, and has the potential to be utilized in transgenic breeding to improve abiotic stress tolerance in crop plants.     

The role of ABA and MAPK signaling pathways in plant abiotic stress responses

As sessile organisms, plants have developed specific mechanisms that allow them to rapidly perceive and respond to stresses in the environment. Among the evolutionarily conserved pathways, the ABA (abscisic acid) signaling pathway has been identified as a central regulator of abiotic stress response in plants, triggering major changes in gene expression and adaptive physiological responses. ABA induces protein kinases of the SnRK family to mediate a number of its responses. Recently, MAPK (mitogen activated protein kinase) cascades have also been shown to be implicated in ABA signaling. Therefore, besides discussing the role of ABA in abiotic stress signaling, we will also summarize the evidence for a role of MAPKs in the context of abiotic stress and ABA signaling.     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.     

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.     

The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

Molecular Cloning and Expression Analysis of Nine ThTrx Genes in Tamarix hispida

Thioredoxins are small conserved proteins that play key roles in the oxidative stress response. In this study, nine Trx genes, including five Trxhs, three Trxms, and one Trx-like gene, were cloned from Tamarix hispida. The roles of these ThTrx genes were investigated under various abiotic stress conditions. Expression profiles of the nine ThTrx genes in response to different abiotic stresses in leaf and root tissues were constructed using quantitative real time-polymerase chain reaction. Differential expression of all nine ThTrx genes was observed (>2-fold) in response to NaCl, PEG, or CdCl₂ stress in at least one tissue, indicating that all of these genes act in abiotic stress responses. All ThTrx genes were induced (>2-fold) by abscisic acid (ABA) treatment in the leaves and especially in the roots, suggesting that ABA-dependent signaling pathways regulate ThTrxs. These results demonstrate that ThTrx expression constitutes an adaptive response to abiotic stress in T. hispida and plays an important role in abiotic stress tolerance.     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

A Novel Role for Arabidopsis CBL1 in Affecting Plant Responses to Glucose and Gibberellin during Germination and Seedling Development

Glucose and phytohormones such as abscisic acid (ABA), ethylene, and gibberellin (GA) coordinately regulate germination and seedling development. However, there is still inadequate evidence to link their molecular roles in affecting plant responses. Calcium acts as a second messenger in a diverse range of signal transduction pathways. As calcium sensors unique to plants, calcineurin B-like (CBL) proteins are well known to modulate abiotic stress responses. In this study, it was found that CBL1 was induced by glucose in Arabidopsis. Loss-of-function mutant cbl1 exhibited hypersensitivity to glucose and paclobutrazol, a GA biosynthetic inhibitor. Several sugar-responsive and GA biosynthetic gene expressions were altered in the cbl1 mutant. CBL1 protein physically interacted with AKINβ1, the regulatory β subunit of the SnRK1 complex which has a central role in sugar signaling. Our results indicate a novel role for CBL1 in modulating responses to glucose and GA signals.     

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1‐related protein kinase 2.10 in response to osmotic stress

SNF1‐related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA‐dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA‐non‐activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S‐segment is involved in the regulation of dehydrin subcellular localization in response to stress.