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Frank Gubler

Publications and source records attributed to Frank Gubler.

13 recordsLinked to original sources
PubMed2006

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase.

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.

Abscisic Acid↗
PubMed2006

Barley (Hordeum vulgare L.).

Crop improvement is limited by the availability of valuable traits in sexually compatible species. Access to new characters using genetic engineering would be of great value. Barley has been transformed using microprojectile bombardment and by direct gene transfer to protoplasts, but neither method has been able to produce fertile transformants in large numbers with simple transgene integration characteristics. Agrobacterium-mediated transformation was first achieved in 1997, and it has become the method of choice. Using immature embryos of the barley variety Golden Promise as the target organ, the binary vector pWBVec8 containing the intron-interrupted hygromycin resistance gene hph as the selectable marker, and selection of transformed cells on hygromycin, the Agrobacterium method is efficient, and the transgene insertion characteristics are superior to other methods. However, the procedure is strongly genotype dependent. In this report, we describe a transformation protocol giving details of plant culture, embryo isolation and preparation, vector details, Agrobacterium culture, infection methods, subsequent procedures for callus generation and plantlet production, and analysis of transgenic plants.

Drug Resistance↗
PubMed2005

The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.

The functions of the vast majority of genes encoding R2R3 MYB domain proteins remain unknown. The closely related MYB33 and MYB65 genes of Arabidopsis thaliana have high sequence similarity to the barley (Hordeum vulgare) GAMYB gene. T-DNA insertional mutants were isolated for both genes, and a myb33 myb65 double mutant was defective in anther development. In myb33 myb65 anthers, the tapetum undergoes hypertrophy at the pollen mother cell stage, resulting in premeiotic abortion of pollen development. However, myb33 myb65 sterility was conditional, where fertility increased both under higher light or lower temperature conditions. Thus, MYB33/MYB65 facilitate, but are not essential for, anther development. Neither single mutant displayed a phenotype, implying that MYB33 and MYB65 are functionally redundant. Consistent with functional redundancy, promoter-beta-glucuronidase (GUS) fusions of MYB33 and MYB65 gave identical expression patterns in flowers (sepals, style, receptacle, anther filaments, and connective but not in anthers themselves), shoot apices, and root tips. By contrast, expression of a MYB33:GUS translational fusion in flowers was solely in young anthers (consistent with the male sterile phenotype), and no staining was seen in shoot meristems or root tips. A microRNA target sequence is present in the MYB genes, and mutating this sequence in the MYB33:GUS fusion results in an expanded expression pattern, in tissues similar to that observed in the promoter-GUS lines, implying that the microRNA target sequence is restricting MYB33 expression. Arabidopsis transformed with MYB33 containing the mutated microRNA target had dramatic pleiotrophic developmental defects, suggesting that restricting MYB33 expression, especially in the shoot apices, is essential for proper plant development.

Alleles↗
PubMed2005

Dormancy release, ABA and pre-harvest sprouting.

Seed dormancy is an adaptive trait that enables the seeds of many species to remain quiescent until conditions become favorable for germination. Dormancy is normally initiated during seed maturation and maintained to seed maturity. In mature seeds, the loss of dormancy may be gradual (after-ripening) or can be terminated by chilling and other environmental triggers. Dormancy is an important trait for many important crop species: it inhibits pre-harvest spouting or vivipary, a widespread problem in many regions of the world. Too much dormancy, however, can lead to non-uniform germination in the field. Recent progress has been made in understanding the role of abscisic acid metabolism and dormancy release in both model plants and crop species. Advances in our understanding of the molecular mechanisms that are involved in dormancy, along with approaches using quantitative genetics, will provide new strategies through which the desired level of dormancy can be introduced into crop species.

Abscisic Acid↗
PubMed2004

Dormancy of Arabidopsis seeds and barley grains can be broken by nitric oxide.

Seeds of Arabidopsis thaliana (L.) Heynh. and grains of barley ( Hordeum vulgare L.) were used to characterize the affects of nitric oxide (NO) on seed dormancy. Seeds of the C24 and Col-1 ecotypes of Arabidopsis are almost completely dormant when freshly harvested, but dormancy was broken by stratification for 3 days at 4 degrees C or by imbibition of seeds with the NO donor sodium nitroprusside (SNP). This effect of SNP on dormancy of Arabidopsis seeds was concentration dependent. SNP concentrations as low as 25 microM reduced dormancy and stimulated germination, but SNP at 250 microM or more impaired seedling development, including root growth, and inhibited germination. Dormancy was also reduced when Arabidopsis seeds were exposed to gases that are generated by solutions of SNP. Nitrate and nitrite, two other oxides of nitrogen, reduced the dormancy of Arabidopsis seeds, but much higher concentrations of these were required compared to SNP. Furthermore, the kinetics of germination were slower for seeds imbibed with either nitrate or nitrite than for seeds imbibed with SNP. Although seeds imbibed with SNP had reduced dormancy, seeds imbibed with SNP and abscisic acid (ABA) remained strongly dormant. This may indicate that the effects of ABA action on germination are downstream of NO action. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (cPTIO) strengthened dormancy of unstratified and briefly stratified Arabidopsis seeds. Dormancy of three cultivars of barley was also reduced by SNP. Furthermore, dormancy in barley grain was strengthened by imbibition of grain with cPTIO. The data presented here support the conclusion that NO is a potent dormancy breaking agent for seeds and grains. Experiments with the NO scavenger suggest that NO is an endogenous regulator of seed dormancy.

Arabidopsis↗
PubMed2004

GMPOZ, a BTB/POZ domain nuclear protein, is a regulator of hormone responsive gene expression in barley aleurone.

GAMYB is a GA-responsive activator of hydrolase gene expression in the aleurone layer of germinated cereal grains. We have isolated a putative GAMYB-binding protein, GMPOZ, which contains a BTB/POZ domain found in certain animal transcriptional regulators. Although BTB/POZ domain proteins are numerous in plants, very few are yet characterized. We found that GMPOZ is nuclear localized and that GMPOZ mRNA is expressed highly in anthers as well as aleurone. Transient silencing of the GMPOZ gene suggests that GMPOZ is involved in hormone responsive gene expression in aleurone.

Amino Acid Sequence↗
PubMed2004

Molecular mechanism of gibberellin signaling in plants.

The hormone gibberellin (GA) plays an important role in modulating diverse processes throughout plant development. In recent years, significant progress has been made in the identification of upstream GA signaling components and trans- and cis-acting factors that regulate downstream GA-responsive genes in higher plants. GA appears to derepress its signaling pathway by inducing proteolysis of GA signaling repressors (the DELLA proteins). Recent evidence indicates that the DELLA proteins are targeted for degradation by an E3 ubiquitin ligase SCF complex through the ubiquitin-26S proteasome pathway.

Arabidopsis↗
PubMed2003

A role for HvGAMYB in anther development.

HvGAMYB is a transcription factor that was first identified in barley aleurone cells and shown to be upregulated by gibberellin (GA). Using RNA and immunoblot analysis we have shown HvGAMYB is also strongly expressed in barley anthers. Transgenic barley expressing a HvGAMYB:GFP fusion gene have been created and GAMYB expression in anthers analysed. GFP expression was clearly visible during early anther development in the nuclei of the epidermis, endothecium, middle layer and tapetum. Expression in the epidermis and endothecium persists until just prior to anther dehiscence, expression in the other two cell layers is visible until they are compressed and broken down as the microspores develop. Further evidence of a role for HvGAMYB in anther development was provided by the creation of transgenic barley over-expressing the HvGAMYB gene. Associated with the increase in HvGAMYB levels was a progressive decrease in anther size, particularly a decrease in anther length. Anthers also became increasingly lighter in colour. Anthers with fourfold more HvGAMYB protein than non-transgenic controls failed to dehisce and were male sterile, anthers with approximately three to fourfold endogenous GAMYB protein levels were smaller and paler but still shed normally. To investigate the hormonal regulation of HvGAMYB expression in anthers, HvGAMYB and SLN1 protein levels in anthers were analysed following application of GA3. As in cereal aleurone, HvGAMYB levels were found to increase and SLN1 levels decrease following GA3 application suggesting a similar GA-signalling pathway to that in aleurone exists in anthers.

Fertility↗
PubMed2003

A Mak-like kinase is a repressor of GAMYB in barley aleurone.

GAMYB is a gibberellin (GA)-regulated activator of hydrolase gene expression in the aleurone layer of germinating cereal grains. Although it is clear that GAMYB expression is regulated by GA, more remains to be understood about how this transcription factor operates within the GA-response pathway. In order to isolate new components from the GA-response pathway, barley aleurone libraries were screened for GAMYB-binding proteins using a recently developed yeast two-hybrid system, which is compatible with the use of transcription factors as baits. We isolated a new member of the emerging Mak-subgroup of cdc2- and MAP kinase-related protein kinases. We have termed this GAMYB-binding protein KGM (for kinase associated with GAMYB). Transient expression of KGM specifically repressed alpha-amylase promoter activity at the level of GAMYB function but a mutation designed to de-stabilise the activation loop of KGM alleviated this repression. We propose that KGM is a negative regulator of GAMYB function in aleurone that may prevent precocious hydrolase gene expression.

Amino Acid Sequence↗
PubMed2003

cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in aleurone cells and inhibits GA-dependent hydrolysis of storage protein and phytate.

The putative H+/Cl- symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when aleurone cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in aleurone layers and prevents synthesis of secretory proteins such as alpha-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the aleurone layer. Vacuolation of the aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl- symporter in aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of alpha-amylase when Cl- ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated cells.

Abscisic Acid↗
PubMed2002

Mutants at the Slender1 locus of barley cv Himalaya. Molecular and physiological characterization.

A dominant dwarf mutant of barley (Hordeum vulgare) that resembles dominant gibberellin (GA) "-insensitive" or "-nonresponsive" mutants in other species is described. alpha-Amylase production by endosperm half-grains of the mutant required GA3 at concentrations about 100 times that of the WT. The mutant showed only a slight growth response to GA3, even at very high concentrations. However, when additionally dwarfed, growth rate responded to GA3 over the normal concentration range, although only back to the original (dwarf) elongation rate. Genetic studies indicated that the dominant dwarf locus was either closely linked or identical to the Sln1 (Slender1) locus. A barley sequence related to Arabidopsis GAI/RGA was isolated, and shown to represent the Sln1 locus by the analysis of sln1 mutants. The dominant dwarf mutant was also altered in this sequence, indicating that it too is an allele at Sln1. Thus, mutations at Sln1 generate plants of radically different phenotypes; either dwarfs that are largely dominant and GA "-insensitive/-nonresponsive," or the recessive slender types in which GA responses appear to be constitutive. Immunoblotting studies showed that in growing leaves, SLN1 protein localized almost exclusively to the leaf elongation zone. In mutants at the Sln1 locus, there were differences in both the abundance and distribution of SLN1 protein, and large changes in the amounts of bioactive GAs, and of their metabolic precursors and catabolites. These results suggest that there are dynamic interactions between SLN1 protein and GA content in determining leaf elongation rate.

Alleles↗
PubMed2002

Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression.

We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of alpha-amylase gene expression, in aleurone cells of barley (Hordeum vulgare). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1 (Slender1) locus, is necessary for repression of GAMYB in barley aleurone cells. The activity of SLN1 in aleurone cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1, and from the gse1 mutant (that affects GA "sensitivity"), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.

Abscisic Acid↗