The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

Escherichia coli xth mutants are hypersensitive to hydrogen peroxide.

Escherichia coli mutants lacking exonuclease III (xthA) are exceptionally sensitive to hydrogen peroxide. They are killed by H2O2 at 20 times the rate of wild-type bacteria and at 3 to 4 times the rate of recA cells. This is the first clear phenotypic sensitivity reported for xth- E. coli and should aid in clarifying peroxide-induced lethality and the in vivo role of exonuclease III.     

Isolation and characterization of novel mutants affecting the abscisic acid sensitivity of Arabidopsis germination and seedling growth

To gain more insight into ABA signaling mechanisms, we conducted genetic screens searching for mutants with altered ABA response in germination and post-germination growth. We isolated seven putative ABA-hypersensitive Arabidopsis mutants and named them ABA-hypersensitive germination (ahg). These mutants exhibited diminished germination or growth ability on medium supplemented with ABA. We further studied four of them: ahg1, ahg2, ahg3 and ahg4. Mapping suggested that they were new ABA-hypersensitive loci. Characterization showed that all of them had enhanced sensitivity to salinity and high osmotic stress in germinating seeds, whereas they each had distinct sugar responses. RT-PCR experiments showed that the expression patterns of the ABA-inducible genes RAB18, AtEm1, AtEm6 and ABI5 in germinating seeds were affected by these four ahg mutations, whereas those of ABI3 and ABI4 were not. ahg4 displayed slightly increased mRNA levels of several ABA-inducible genes upon ABA treatment. By contrast, ahg1 had no clear ABA-hypersensitive phenotypes in adult plants despite its strong phenotype in germination. These results suggest that ahg1, ahg2, ahg3 and ahg4 are novel ABA-hypersensitive mutants representing distinct components in the ABA response.     

Seed dormancy and germination: the role of abscisic acid and gibberellins and the importance of hormone mutants

Over the past decades many studies have aimed at elucidating the regulation of seed dormancy and germination. Many hypotheses have been proposed and rejected but the regulatory principle behind changes in dormancy and induction of germination is still a black box. The majority of proposed mechanisms have a role for certain plant hormones in common. Abscisic acid and the gibberellins are the hormones most frequently suggested to control these processes. The development of hormone-deficient mutants made it possible to provide direct evidence for the involvement of hormones in germination and dormancy related processes.In the present paper an attempt is made to assess the role of abscisic acid and gibberellins in the transitions between dormant and non-dormant states and germination. First a conceptual framework is presented in which the different states of dormancy and germination are defined in order to contribute to a solution of the semantic confusion about these terms that has existed since the beginning of seed physiology.It is concluded that abscisic acid plays a pivotal role during the development of primary dormancy and gibberellins are involved in the induction of germination. Changes in sensitivity to these hormones occur during changes in dormancy. Both synthesis of and responsiveness to the hormones are controlled by natural environmental factors such as light, temperature and nitrate.     

sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.     

Evidence of a role for tyrosine dephosphorylation in the control of postgermination arrest of development by abscisic acid in Arabidopsis thaliana L

In the present paper evidence is presented indicating that tyrosine dephosphorylation is a key regulatory mechanism in postgermination arrest of Arabidopsis thaliana L. seed development mediated by abscisic acid (ABA). By using phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, the sensitivity to the inhibitory effect of ABA on seed germination is enhanced. Consistent with this finding, we demonstrate that the ABA-responsive gene, RAB18, is hyperinduced in seeds imbibed in ABA plus PAO, compared with seeds imbibed only with ABA.     

Ascorbic acid and reactive oxygen species are involved in the inhibition of seed germination by abscisic acid in rice seeds

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.     

Phenotypic interactions between abscisic acid deficient tomato mutants

Summary A series of double mutant homozygotes have been produced from three wilty tomato mutants; flacca, sitiens and notabilis. The phenotypic interaction between the mutant genes has been studied. The severity of phenotype in the double mutants does not correspond to that predicted from the single mutant homozygotes. The results are discussed in relation to the probable involvement of the mutants in abscisic acid metabolism.     

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.     

Deoxycytidine kinase-deficient mutants of Chinese hamster ovary cells are hypersensitive to DNA alkylating agents

Chinese hamster ovary cell strains deficient in deoxycytidine kinase activity were selected by isolating mutants resistant to high concentrations of the analogue arabinosyl cytosine. Mutants isolated were deficient in the pool of dCTP, supporting earlier a suggestion that the deoxycytidine kinase may play a role in the turnover and maintenance of the dCTP pool. Consistent with earlier observations that increased intracellular levels of dTTP relative to dCTP lead to increased sensitivy to monofunctional DNA alkylating agents, deoxycytidine kinase-deficient mutants showed a 2–5-fold increase in sensitivity to the cytotoxic and mutagenic effects of one agent, ethyl methanesulfonate (EMS). The survival of the two kinase-deficient strains after mutagen treatment was clearly related to dCTP level as the strain with lowest dCTP was most sensitive to EMS. Thus hypersensitivity to this class of DNA damaging agents can result from cellular mutations decreasing the intracellular level of dCTP.     

Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and abscisic acid during the development and germination of barley seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca(2+)-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.     

Pseudomonas putida mutants in the exbBexbDtonB gene cluster are hypersensitive to environmental and chemical stressors

The genes in the exbBexbDtonB cluster of Pseudomonas putida DOT-T1E are co-transcribed. We have generated non-polar mutants in each of the genes by inserting an aphA3 cassette encoding kanamycin resistance. All three mutants show similar phenotypes: the mutants are unable to grow on minimal medium under iron deficiency conditions. Furthermore, regardless of iron conditions, all mutants are hypersensitive to antibiotics, p-hydroxybenzoate and toluene, chemicals that are extruded from the cell by efflux pumps. These findings are discussed in terms of the involvement of the TonB system in the energization of outer membrane functions necessary for the import or export of different compounds in P. putida.     

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.     

Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.     

The reciprocal regulation of abscisic acid and ethylene biosyntheses

Ethylene and abscisic acid (ABA) have compact effects on plant development and stress responses. It is not well understood about the mechanism of ABA modulation in ethylene biosynthesis. In our recent research, HY5-AtERF11 regulon was evidenced to connect the ABA action and ethylene biosynthesis. In this paper, by analyzing the expression of ABA biosynthesis genes and the ABA concentration in ethylene over-production mutants, we demonstrated that ethylene production affected by HY5-AtERF11 regulon targeted gene increased the expression of ABA biosynthesis genes and its contents. In addition, we discussed that HY5 might function as a convergence point of multiple hormones in response to light.     

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.     

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.