Responses of wild type and abscisic acid mutants ofArabidopsis thaliana to cadmium

Heavy metal-induced inhibitory effects are reported to be concomitant with an increase in endogenous abscisic acid (ABA) levels in plant tissues indicating the possibility of this phytohormone mediating a part of the metal-imposed phytotoxicity. We examined this possibility by comparing the seed germination and seedling growth responses of ABA-deficient (aba-1, aba-3 and aba-4) and ABA-insensitive (abi2-1 and abi3-1) mutants of Arabidopsis thaliana to Cd with those of the wild type (Landsberg erecta, Ler). Assuming that Cd imposed a part of its toxic influence via affecting a rise in endogenous ABA level, all ABA mutants studied could be predicted to exhibit reduced responsiveness to Cd exposure in comparison to the wild type. However, the data obtained both in germination and growth assays were not consistent with this prediction. In germination assays, all ABA mutants proved consistently more sensitive than the wild type to Cd. In case of growth (root length and seedling fresh weight), the magnitude of Cd-induced inhibition in ABA mutants (aba-1, abi2-1 and abi3-1) was generally comparable to that in the wild type. Based on these observations a direct mediatory role of ABA in Cd-imposed phytotoxic effects on early growth could be excluded. The possible significance of heavy metal-dependent increase in endogenous ABA levels in plant tissues is discussed.     

TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity

In plants, clade A type 2C protein phosphatases (PP2CAs) have emerged as major players in abscisic acid (ABA)-regulated stress responses by inhibiting protein kinase activity. However, how different internal and external environmental signals modulate the activity of PP2CAs are not well known. The transmembrane kinase (TMK) protein 4 (TMK4), one member of a previously identified receptor kinase subfamily on the plasma membrane that plays vital roles in plant cell growth, directly interacts with PP2CAs member (ABA-Insensitive 2, ABI2). tmk4 mutant is hypersensitive to ABA in both ABA-inhibited seed germination and primary root growth, indicating that TMK4 is a negative regulator in ABA signaling pathway. Further analyses indicate that TMK4 phosphorylates ABI2 at three conserved Ser residues, thus enhancing the activity of ABI2. The phosphorylation-mimic ABI2^{S139DS140DS266D} can complement but non-phosphorylated form ABI2^{S139AS140AS266A} cannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.     

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.     

ZmOST1 mediates abscisic acid regulation of guard cell ion channels and drought stress responses

The phytohormone abscisic acid (ABA) is an important mediator in the drought response, participating in, among other processes, stomatal movements. In Arabidopsis thaliana, the serine/threonine protein kinase, OST1, regulates this response, but the function of its maize homolog has yet to be established. Here, we isolated ZmOST1 and show that its encoded protein indeed acts to regulate guard cell movement. ZmOST1 was ubiquitously expressed throughout the plant, being highly expressed in guard cells, and inducible both by exogenous ABA and water stress. Transient expression of a ZmOST1-GFP fusion protein, in maize mesophyll protoplasts, indicated its subcellular localization in the cytoplasm and nucleus. A Zmost1 loss-of-function mutant exhibited reduced sensitivity to ABA-activated slow anion channels in maize guard cells, and reduced drought tolerance. Constitutive expression of ZmOST1, in an A. thaliana ost1-1 mutant rescued the phenotype with respect both to the sensitivity of guard cell slow anion currents to ABA treatment and stomatal closure. Our findings indicate a positive regulatory role for ZmOST1 in guard cell ABA signaling and drought response in maize plants.     

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.     

Interactions between abscisic acid and ethylene signaling cascades

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.     

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO(2), suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H(2)O(2) or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.     

Disruption of a guard cell-expressed protein phosphatase 2A regulatory subunit,RCN1, confers abscisic acid insensitivity in Arabidopsis

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell-expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca(2+) increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling.     

Arabidopsis CPR5 independently regulates seed germination and postgermination arrest of development through LOX pathway and ABA signaling

The phytohormone abscisic acid (ABA) and the lipoxygenases (LOXs) pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently.     

RING-box proteins regulate leaf senescence and stomatal closure via repression of ABA transporter gene ABCG40

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.     

Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1-2

Cytosolic calcium increases were analyzed in guard cells of the Arabidopsis farnesyltransferase deletion mutant era1-2 (enhanced response to abscisic acid). At low abscisic acid (ABA) concentrations (0.1 microM), increases of guard cell cytosolic calcium and stomatal closure were activated to a greater extent in the era1-2 mutant compared with the wild type. Patch clamping of era1-2 guard cells showed enhanced ABA sensitivity of plasma membrane calcium channel currents. These data indicate that the ERA1 farnesyltransferase targets a negative regulator of ABA signaling that acts between the points of ABA perception and the activation of plasma membrane calcium influx channels. Experimental increases of cytosolic calcium showed that the activation of S-type anion currents downstream of cytosolic calcium and extracellular calcium-induced stomatal closure were unaffected in era1-2, further supporting the positioning of era1-2 upstream of cytosolic calcium in the guard cell ABA signaling cascade. Moreover, the suppression of ABA-induced calcium increases in guard cells by the dominant protein phosphatase 2C mutant abi2-1 was rescued partially in era1-2 abi2-1 double mutant guard cells, further reinforcing the notion that ERA1 functions upstream of cytosolic calcium and indicating the genetic interaction of these two mutations upstream of ABA-induced calcium increases.     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.