Characterization of two new channel protein genes in Arabidopsis.
Aquaporins, small channel proteins, found in a variety of organisms are members of the major intrinsic protein (MIP) superfamily and have been shown to facilitate water transport when expressed in Xenopus oocytes. We isolated two Arabidopsis cDNAs, SIMIP and SITIP, that encode protein homologues of the MIP superfamily. SIMIP exhibits a high degree of sequence homology to PIP3 and MIP1, and thus may belong to the plasmamembrane intrinsic protein (PIP) subfamily, whereas salt-stress inducible tonoplast intrinsic protein (SITIP) is highly homologous to VM23 and gamma-TIP, and therefore may belong to the TIP subfamily. Expression studies revealed that the two genes showed a different expression pattern. The SIMIP gene was expressed in a tissue-specific manner, for example, its highest transcript level is found in flowers, relatively low levels in siliques, and very low level in leaves and roots. In contrast, SITIP was expressed in nearly equal amounts in all the tissues we examined. Also, the expression of SIMIP and SITIP showed a temporal regulation pattern. For example, the highest expression level was at 1 week after germination. In addition, the transcript levels of SIMIP and SMTIP were increased upon NaCl and ABA treatments. The biological function of the 2 genes were investigated using two NaCl stress-sensitive yeast mutant strains. The mutant yeast cells expressing these 2 genes were more resistant to high NaCl conditions. The results suggest that the proteins encoded by these genes may be involved in the osmoregulation in plants under high osmotic stress such as under a high salt condition. (+info)
An Arabidopsis GSK3/shaggy-like gene that complements yeast salt stress-sensitive mutants is induced by NaCl and abscisic acid.
GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis. (+info)
A cluster of ABA-regulated genes on Arabidopsis thaliana BAC T07M07.
Arabidopsis thaliana BAC T07M07 encoding the abscisic acid-insensitive 4 (ABI4) locus has been sequenced completely. It contains a 95,713-bp insert and 24 predicted genes. Most putative genes were confirmed by gel-based RNA profiling and a cluster of ABA-regulated genes was identified. One of the 24 genes, designated PP2C5, encodes a putative protein phosphatase 2C. The encoded protein was expressed in Escherichia coli, and its enzyme activity in vitro was confirmed. (+info)
Flow cytometry and surface plasmon resonance analyses demonstrate that the monoclonal antibody JIM19 interacts with a rice cell surface component involved in abscisic acid signalling in protoplasts.
Abscisic acid (ABA) is a plant hormone involved in many developmental and physiological processes, but as yet, no ABA receptor has been identified. Flow cytometry of rice protoplasts and immunoblotting of purified plasma membranes (PMs) have been used to demonstrate that the monoclonal antibody JIM19 recognizes carbohydrate epitopes of cell surface glycoproteins. Using surface plasmon resonance technology specific binding of PMs to JIM19 was observed. Such interaction was antagonized significantly by ABA, but not by the biologically inactive ABA catabolite phaseic acid. These in vitro interactions were correlated with the biological activities of JIM19, ABA and phaseic acid on activation of the ABA-inducible Em promoter using two different transient reporter gene assays, beta-glucuronidase/luciferase and quantitative flow cytometry of Aequoria green fluorescent protein. Pre-treatment with JIM19 resulted in significant inhibition of ABA-inducible gene expression. Taken together, these data suggest that JIM19 interacts with a functional PM complex involved in ABA signalling. (+info)
Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.
Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt). (+info)
ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.
The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses. (+info)
Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis.
Sucrose synthase (Sus) is a key enzyme of sucrose metabolism. Two Sus-encoding genes (Sus1 and Sus2) from Arabidopsis thaliana were found to be profoundly and differentially regulated in leaves exposed to environmental stresses (cold stress, drought or O(2) deficiency). Transcript levels of Sus1 increased on exposure to cold and drought, whereas Sus2 mRNA was induced specifically by O(2) deficiency. Both cold and drought exposures induced the accumulation of soluble sugars and caused a decrease in leaf osmotic potential, whereas O(2) deficiency was characterized by a nearly complete depletion in sugars. Feeding abscisic acid (ABA) to detached leaves or subjecting Arabidopsis ABA-deficient mutants to cold stress conditions had no effect on the expression profiles of Sus1 or Sus2, whereas feeding metabolizable sugars (sucrose or glucose) or non-metabolizable osmotica [poly(ethylene glycol), sorbitol or mannitol] mimicked the effects of osmotic stress on Sus1 expression in detached leaves. By using various sucrose/mannitol solutions, we demonstrated that Sus1 was up-regulated by a decrease in leaf osmotic potential rather than an increase in sucrose concentration itself. We suggest that Sus1 expression is regulated via an ABA-independent signal transduction pathway that is related to the perception of a decrease in leaf osmotic potential during stresses. In contrast, the expression of Sus2 was independent of sugar/osmoticum effects, suggesting the involvement of a signal transduction mechanism distinct from that regulating Sus1 expression. The differential stress-responsive regulation of Sus genes in leaves might represent part of a general cellular response to the allocation of carbohydrates during acclimation processes. (+info)
A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription.
The transcription factor VP1 regulates maturation and dormancy in plant seeds by activating genes responsive to the stress hormone abscisic acid (ABA). Although activation involves ABA-responsive elements (ABREs), VP1 itself does not specifically bind ABREs. Instead, we have identified and cloned a basic region leucine zipper (bZIP) factor, TRAB1, that interacts with both VP1 and ABREs. Transcription from a chimeric promoter with GAL4-binding sites was ABA-inducible if cells expressed a GAL4 DNA-binding domain::TRAB1 fusion protein. Results indicate that TRAB1 is a true trans-acting factor involved in ABA-regulated transcription and reveal a molecular mechanism for the VP1-dependent, ABA-inducible transcription that controls maturation and dormancy in plant embryos. (+info)