Regulation and function of Arabidopsis AtGALK2 gene in abscisic acid response signaling

AtGALK2 belongs to galactokinase of GHMP family in Arabidopsis thaliana. Two homozygous T-DNA insertion mutants (Atgalk2-1 and Atgalk2-2) of the AtGALK2 gene were identified. The AtGALK2 gene was highly expressed in flowers and roots, but less in stems, leaves and petioles. It was found that the expression of AtGALK2 gene was induced by NaCl and ABA. The two Atgalk2 mutants showed higher germination activity when treated with ABA and NaCl than wild type (Col-0). Through comparing the results of seed germination, root growth, stomatal aperture, water loss, and proline accumulation between the Atgalk2 mutants and Col-0, it was found that Atgalk2 mutants showed less sensitive to ABA than Col-0. The expression levels of ABI1, ABI2, RAB18, ABF3, RD22, RD29A, and RD29B in the Atgalk2 mutants were higher than in Col-0. However, the expression level of OST1 in the Atgalk2 mutants was lower than in Col-0. Taken together, these results suggested AtGALK2 was required for abscisic acid regulation of seed germination, root growth and gene expression, and was involved in salt and osmotic stress response in the early development stage. This study provides important clues to galactokinase activities of GHMP family in ABA signaling and plant development.     

Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses

Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways.     

Arabidopsis acyl‐CoA‐binding protein ACBP1 participates in the regulation of seed germination and seedling development

A family of six genes encoding acyl‐CoA‐binding proteins (ACBPs), ACBP1–ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1‐over‐expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over‐production in 12‐day‐old seedlings up‐regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress‐responsive genes: ABA‐RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH‐TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12‐day‐old seedlings of ACBP1‐over‐expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two‐hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA‐mediated seed germination and seedling development.     

GPT2: a glucose 6-phosphate/phosphate translocator with a novel role in the regulation of sugar signalling during seedling development

Background and Aims

GPT2, a glucose 6-phosphate/phosphate translocator, plays an important role in environmental sensing in mature leaves of Arabidopsis thaliana. Its expression has also been detected in arabidopsis seeds and seedlings. In order to examine the role of this protein early in development, germination and seedling growth were studied.

Methods

Germination, greening and establishment of seedlings were monitored in both wild-type Arabidopsis thaliana and in a gpt2 T-DNA insertion knockout line. Seeds were sown on agar plates in the presence or absence of glucose and abscisic acid. Relative expression of GPT2 in seedlings was measured using quantitative PCR.

Key Results

Plants lacking GPT2 expression were delayed (25–40 %) in seedling establishment, specifically in the process of cotyledon greening (rather than germination). This phenotype could not be rescued by glucose in the growth medium, with greening being hypersensitive to glucose. Germination itself was, however, hyposensitive to glucose in the gpt2 mutant.

Conclusions

The expression of GPT2 modulates seedling development and plays a crucial role in determining the response of seedlings to exogenous sugars during their establishment. This allows us to conclude that endogenous sugar signals function in controlling germination and the transition from heterotrophic to autotrophic growth, and that the partitioning of glucose 6-phosphate, or related metabolites, between the cytosol and the plastid modulates these developmental responses.     

Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis

Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought. Bas J. W. Dekkers and Jolanda A. M. J. Schuurmans contributed equally to this paper.     

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.     

Arabidopsis EIN2 modulates stress response through abscisic acid response pathway

The nuclear protein ETHYLENE INSENSITIVE2 (EIN2) is a central component of the ethylene signal transduction pathway in plants, and plays an important role in mediating cross-links between several hormone response pathways, including abscisic acid (ABA). ABA mediates stress responses in plants, but there is no report on the role of EIN2 on plant response to salt and osmotic stresses. Here, we show that EIN2 gene regulates plant response to osmotic and salt stress through an ABA-dependent pathway in Arabidopsis. The expression of the EIN2 gene is down-regulated by salt and osmotic stress. An Arabidopsis EIN2 null mutant was supersensitive to both salt and osmotic stress conditions. Disruption of EIN2 specifically altered the expression pattern of stress marker gene RD29B in response to the stresses, but not the stress- or ABA-responsive genes RD29A and RD22, suggesting EIN2 modulates plant stress responses through the RD29B branch of the ABA response. Furthermore, disruption of EIN2 caused substantial increase in ABA. Lastly, our data showed that mutations of other key genes in ethylene pathway also had altered sensitivity to abiotic stresses, indicating that the intact ethylene may involve in the stress response. Taken together, the results identified EIN2 as a cross-link node in ethylene, ABA and stress signaling pathways, and EIN2 is necessary to induce developmental arrest during seed germination, and seedling establishment, as well as subsequent vegetative growth, thereby allowing the survival and growth of plants under the adverse environmental conditions. Youning Wang and Chuang Liu contributed equally to this work.     

Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth

Summary. Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dα (PLDα) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms. Correspondence and reprints: Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, U.S.A.     

Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens

Plant hormone abscisic acid (ABA) is found in a wide range of land plants, from mosses to angiosperms. However, our knowledge concerning the function of ABA is limited to some angiosperm plant species. We have shown that the basal land plant Physcomitrella patens and the model plant Arabidopsis thaliana share a conserved abscisic acid (ABA) signaling pathway mediated through ABI1-related type 2C protein phosphatases (PP2Cs). Ectopic expression of Arabidopsis abi1-1, a dominant allele of ABI1 that functions as a negative regulator of ABA signaling, or targeted disruption of Physcomitrella ABI1-related gene (PpABI1A) resulted in altered ABA sensitivity and abiotic stress tolerance of Physcomitrella, as demonstrated by osmostress and freezing stress. Moreover, transgenic Physcomitrella overexpressing abi1-1 showed altered morphogenesis. These trangenic plants had longer stem lengths compared to the wild type, and continuous growth of archegonia (female organ) with few sporophytes under non-stress conditions. Our results suggest that PP2C-mediated ABA signaling is involved in both the abiotic stress responses and developmental regulation of Physcomitrella.Key words: ABA, ABI1, Physcomitrella patens, PP2C, signaling