Differential stress responses of early salt-stress responding genes in common wheat

Four early salt-stress responding genes (WESR1-4) in common wheat (Triticum aestivum L.) were analyzed for their temporal accumulation of mRNA during salt stress, osmotic stress and abscisic acid (ABA) treatment. All genes showed transient stimulation by 0.15 M NaCl treatment. WESR1 and WESR2 were induced by both osmotic stress and exogenous ABA treatment. WESR3 responded to exogenous ABA, but not to osmotic stress. WESR4 did not show significant response to either osmotic stress or exogenous ABA treatment. These results suggest that wheat has at least two salt stress signal transduction pathways, an ABA-dependent and ABA-independent pathway.     

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.     

Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid

In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.     

Drought tolerance, abscisic acid and electrolyte leakage in fast-and slow-growing black spruce (Picea mariana) progenies

To investigate the role that drought tolerance plays in growth, abscisic acid (ABA) accumulation and electrolyte leakage during water stress were compared in fast- and slow-growing black spruce ( Picea mariana [Mill.] B. S. P.) progenies. Changes in the ABA content of the needles were quantified using an indirect enzyme-linked immuno-sorbent assay validated by gas chromatography electron capture detection. Needle electrolyte leakage was estimated using a conductivity bridge. Seedlings were stressed using (1) osmotic stress, induced by a stepwise increase in concentrations of polyethylene glycol 3 350 (PEG) for ABA study and (2) air drying for electrolyte leakage study. Progenies did not differ in ABA levels under unstressed conditions, but progeny differences were observed under osmotic stress. Needle ABA content increased up to 500% under osmotic stress. Slow-growing black spruce progenies (25 and 46) accumulated more ABA under moderate (18% PEG), but not severe (25% PEG), osmotic stress. The slow-growing progenies also leaked more electrolytes under moderate to severe water stress and lost 50% electrolytes at a higher xylem tension, suggesting they suffered more injury and were less dehydration tolerant. Our previously-published results showed that slow-growing progenies lost their photosynthesis and stomatal conductance more quickly during osmotic stress and recovered more slowly after rehydration. Therefore, tolerance of dehydration leading to a maintenance of physiological integrity during drought stress could explain the fast growth rates of more vigorous black spruce progenies.     

Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.     

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.     

The soybean root‐specific protein kinase GmWNK1 regulates stress‐responsive ABA signaling on the root system architecture

In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re‐absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root‐specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down‐regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8′‐hydroxylase that functions in ABA catabolism. Furthermore, 35S‐GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild‐type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine‐tune ABA‐dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA‐dependent mechanism distinct from known ABA signaling pathways.     

Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid （ABA） in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction. Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser （GRGDS）, which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-Iike proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.     

渗透胁迫和外源脱落酸对梭梭幼苗生理特性的影响

水分、渗透胁迫和外源脱落酸（ＡＢＡ）对旱生植物梭梭幼苗的某些生理特性存在显著影响。梭梭幼苗有很强的吸水能力,渗透胁迫下脯氨酸含量增加,其细胞膜相对透性对渗透胁迫不敏感,在脱水过程中具有较强的持水能力,外源ＡＢＡ加强了梭梭幼苗的抗渗透胁迫和抗脱水的能力。     

ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis

Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis: The identity of ABA-activated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABA-inducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.     

The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.     

Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: Inverse correlation between leaf abscisic acid accumulation and leaf water loss

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in both abscisic acid (ABA) signaling and stress-induced ABA accumulation. However, little is known about their physiological roles in the whole plant. In this study, the effects of NO and ROS on leaf water control and the roles of ABA were determined using wheat (Triticum aestivum L.) seedlings. As compared with the control, osmotic stress reduced leaf water loss (LWL) while it increased leaf ABA content. The effects of osmotic stress on LWL and ABA contents were partially reversed by NO scavengers or NO synthase (NOS) inhibitors. Furthermore, sodium nitroprusside (SNP) at concentrations between 0.01 and 10 mM all reduced LWL efficiently and induced ABA accumulation in a dose-dependent manner. When ABA synthesis was inhibited by fluridone or actidione, the effects of SNP on LWL were partially reversed. These results suggest that NO is involved in leaf water maintenance of wheat seedlings under osmotic stress, and one of the possible mechanisms is by stimulating ABA synthesis. The ROS scavengers used in our experiments had no effects on either LWL or ABA accumulation induced by osmotic stress. However, all ROS induced LWL reduction and ABA accumulation significantly. Hydrogen peroxide had the same effects as SNP on LWL and induced ABA accumulation in a dose-dependent manner but had a maximal effect at 1 mM. Fluridone reversed the effects of H₂O₂ on both LWL reduction and ABA accumulation, while actidione had no effect. These results suggest that ROS are also involved in leaf water maintenance of wheat seedlings by stimulating ABA biosynthesis, but with a different mechanism to that of NO. The ABA-independent mechanism in NO/ROS regulation of leaf water balance is discussed, in relation to our results.     

Tomato tos1 mutation identifies a gene essential for osmotic tolerance and abscisic acid sensitivity

Osmotic stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify plant genes that are required for osmotic stress tolerance in tomato. As a result, we have isolated a novel mutant in tomato (tos1) caused by a single recessive nuclear mutation that is hypersensitive to general osmotic stress. Growth measurements demonstrated that the tos1 mutant is less sensitive to intracellular abscisic acid (ABA) and this decreased ABA sensitivity of tos1 is a basic cellular trait expressed by the mutant at all developmental stages analysed. It is not caused by a deficiency in the synthesis of ABA because the tos1 seedlings accumulated more ABA than the wild type (WT) after osmotic stress. In contrast, the tss2 tomato mutant, which is also hypersensitive to osmotic stress, is hypersensitive to exogenous ABA. Comparative analysis of tos1 and tss2 indicates that appropriate ABA perception and signalling is essential for osmotic tolerance.