Abscisic acid-insensitive mutations provide evidence for stage-specific signal pathways regulating expression of an Arabidopsis late embryo genesis-abundant (lea) gene

An Arabidopsis homolog of the abscisic acid (ABA)-inducible cotton D19 and wheat Em genes was cloned and its expression assayed at two developmental stages in wild-type, ABA-deficient (aba) and three ABA-insensitive (abi) lines of Arabidopsis thaliana. Expression of this gene was reduced slightly in seeds of aba mutants and approximately ten-fold in abi3 mutants, but seed expression was not decreased in either abi1 or abi2 monogenic mutants. In contrast, the abi1 and abi2 mutants showed a very slight reduction of ABA inducibility in 8-day-old plants, while the responses of aba and abi3 mutants were comparable to that of wild type. Although previous studies have shown that none of the abi mutations show completely stage-specific effects, the results reported here indicate that the importance of each of the ABI loci in regulating this single gene is stage-dependent. Furthermore, the fact that none of the abi mutations show more than minor effects on exogenous ABA inducibility of the Arabidopsis D19/Em homolog in young plants suggests that an additional ABA signalling pathway may be operating during vegetative growth.     

Interactions of abscisic acid and sugar signalling in the regulation of leaf senescence

Leaf senescence can be triggered by a high availability of carbon relative to nitrogen or by external application of abscisic acid (ABA). Most Arabidopsis mutants with decreased sugar sensitivity during early plant development are either ABA insensitive (abi mutants) or ABA deficient (aba mutants). To analyse the interactions of carbon, nitrogen and ABA in the regulation of senescence, wild-type Arabidopsis thaliana (L.) Heynh. and aba and abi mutants were grown on medium with varied glucose and nitrogen supply. On medium containing glucose in combination with low, but not in combination with high nitrogen supply, senescence was accelerated and sucrose, glucose and fructose accumulated strongly. In abi mutants that are not affected in sugar responses during early development (abi1-1 and abi2-1), we observed no difference in the sugar-dependent regulation of senescence compared to wild-type plants. Similarly, senescence was not affected in the sugar-insensitive abi4-1 mutant. In contrast, the abi5-1 mutant did exhibit a delay in senescence compared to its wild type. As ABA has been reported to induce senescence and ABA deficiency results in sugar insensitivity during early development, we expected senescence to be delayed in aba mutants. However, the aba1-1 and aba2-1 mutants showed accelerated senescence compared to their wild types on glucose-containing medium. Our results show that, in contrast to sugar signalling in seedlings, ABA is not required for the sugar-dependent induction of leaf senescence. Instead, increased sensitivity to osmotic stress could have triggered early senescence in the aba mutants.Abbreviations ABA Abscisic acid - aba Abscisic acid deficient - abi Abscisic acid insensitive - F_v/F_m Maximum efficiency of photosystem II photochemistry     

Abscisic acid-insensitive mutations provide evidence for stage-specific signal pathways regulating expression of an Arabidopsis late embryo genesis-abundant (lea) gene

An Arabidopsis homolog of the abscisic acid (ABA)-inducible cotton D19 and wheat Em genes was cloned and its expression assayed at two developmental stages in wild-type, ABA-deficient (aba) and three ABA-insensitive (abi) lines of Arabidopsis thaliana. Expression of this gene was reduced slightly in seeds of aba mutants and approximately ten-fold in abi3 mutants, but seed expression was not decreased in either abi1 or abi2 monogenic mutants. In contrast, the abi1 and abi2 mutants showed a very slight reduction of ABA inducibility in 8-day-old plants, while the responses of aba and abi3 mutants were comparable to that of wild type. Although previous studies have shown that none of the abi mutations show completely stage-specific effects, the results reported here indicate that the importance of each of the ABI loci in regulating this single gene is stage-dependent. Furthermore, the fact that none of the abi mutations show more than minor effects on exogenous ABA inducibility of the Arabidopsis D19/Em homolog in young plants suggests that an additional ABA signalling pathway may be operating during vegetative growth.     

Phosphate deficiency-dependent upregulation of UDP-glucose pyrophosphorylase genes is insensitive to ABA and ethylene status in Arabidopsis leaves

The effects of inorganic phosphate (Pi) deficiency and ABA/ethylene status on expression of UDP-glucose pyrophosphorylase (UGPase) genes (Ugp), involved in sucrose/polysaccharide metabolism, were investigated. Both wild-type (wt), aba and abi mutants (ABA-deficient and -in-sensitive), etr, ein and eto (ethylene resistant and overproducing) grown on Pi-deficient and complete nutrient solution, as well as phol (Pi-deficient) mutants of Arabidopsis thaliana were used for experiments. Generally, Pi-deficiency conditions (including mannose feeding to decrease cytosolic Pi pool) resulted in an increase of Ugp expression in the leaves, under all experimental conditions. Mutant backgrounds reflecting differences in ABA or ethylene status/ sensitivity had no effect on the level of Ugp up-regulation by Pi-stress. Furthermore, feeding ABA to the leaves of wt and pho1 plants had no effect on Ugp expression, regardless of the sucrose status in the leaves. The data suggest that Pi deficiency leading to up-regulation of Ugp acts independently of ABA and ethylene status.     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession Numbers: PpABI1A-AB369256, PpABI1B-AB369255, pphn39k21-AB369257.     

The Role of ABI3 and FUS3 Loci in Arabidopsis thaliana on Phase Transition from Late Embryo Development to Germination

Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

The Ascorbate-deficient vtc-1 Arabidopsis Mutant Shows Altered ABA Accumulation in Leaves and Chloroplasts

Abscisic acid (ABA) accumulation has been analyzed in irrigated and water-stressed wild-type and the vtc-1 mutant of Arabidopsis thaliana, which shows an ascorbate deficiency in leaves of approximately 60%. The amounts of ABA increased progressively up to 2.3-fold in water-stressed wild-type plants, whereas levels were kept at low levels in the irrigated plants. In contrast, initial increases followed by a sharp decrease of abscisic acid levels were observed in water-stressed vtc-1 mutants. Furthermore, the levels of this phytohormone increased up to fivefold in irrigated mutants. This differential accumulation of ABA in the mutant strongly correlated with the ascorbate redox state, but not with ascorbate levels. Changes in ABA levels in leaves paralleled those of chloroplasts. Immunolocalization studies showed a differential ABA accumulation in chloroplasts of vtc-1 mutants, which displayed the highest ABA labeling in irrigated plants. Our results indicate an altered pattern of ABA accumulation in the vtc-1 mutant compared to the wild type, under both irrigated conditions and water-stress conditions, which is strongly dependent on the ascorbate redox state.     

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.     

Identification of further <Emphasis Type="Italic">Craterostigma plantagineum cdt</Emphasis> mutants affected in abscisic acid mediated desiccation tolerance

The resurrection plant (Craterostigma plantagineum) is desiccation tolerant. However, callus derived from this plant, when propagated in vitro, requires exogenously applied abscisic acid (ABA) in order to survive desiccation. Treatment of callus tissue with ABA induces most of the genes that are induced by dehydration in the whole plant. This property has been exploited for the isolation of mutants that show dominant phenotypes resulting from the ectopic expression of endogenous genes induced by the insertion of a foreign promoter. Here we describe new T-DNA tagged Craterostigma desiccation-tolerant (cdt) mutants with different molecular and physiological characteristics, suggesting that different pathways of desiccation tolerance are affected. One of the mutants, cdt-2, constitutively expresses known osmoprotective Lea genes in callus and leaf tissue. Further analysis of this mutant revealed that the tagged locus is similar to a previously characterised gene, CDT-1, which codes for a signalling molecule that confers desiccation tolerance. The nature of the T-DNA insertion provides insight into the mechanism by which the CDT-1/2 gene family functions in ABA signal transduction.     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

The responses of wild-type and ABA mutant Arabidopsis thaliana plants to phosphorus starvation

The growth patterns of plants subjected to phosphorus starvation resemble those caused by treatment with ABA, suggesting that ABA could mediate the response of the plant to phosphorus starvation. We examined the role of ABA in phosphorus stress by comparing growth and biochemical responses of Arabidopsis thaliana ABA mutants aba-1 and abi2-1 to those of wild-type plants. We first characterized acid phosphatase production of wild-type Arabidopsis in response to phosphorus starvation. We found that several acid phosphatase isozymes are present in roots and shoots, but only a subset of these isozymes are induced by phosphorus stress, and they are induced in both organs. Production of acid phosphatase in response to phosphorus stress was not affected by the aba-1 or abi2-1 mutations. Low phosphorus also resulted in decreased growth of both wild-type and ABA mutant plants, and the root-to-shoot ratio was increased in both wild type and mutants. Anthocyanins accumulated in response to phosphorus stress in both wild-type and mutant plants, but the increase was reduced in the aba-1 mutant. Thus, two different ABA mutants responded normally in most respects to phosphorus stress. Our data do not support a major role for ABA in coordinating the phosphorus-stress response.     

A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele

To investigate the molecular mechanisms controlling the process of cold acclimation and to identify genes involved in plant freezing tolerance, mutations that impaired the cold acclimation capability of Arabidopsis thaliana (L.) Heynh. were screened for. A new mutation, frs1 (freezing sensitive 1), that reduced both the constitutive freezing tolerance as well as the freezing tolerance of Arabidopsis after cold acclimation was characterized. This mutation also produced a wilty phenotype and excessive water loss. Plants with the frs1 mutation recovered their wild-type phenotype, their capability to tolerate freezing temperatures and their capability to retain water after an exogenous abscisic acid (ABA) treatment. Measurements of ABA revealed that frs1 mutants were ABA deficient, and complementation tests indicated that frs1 mutation was a new allele of the ABA3 locus showing that a mutation in this locus leads to an impairment of freezing tolerance. These results constitute the first report showing that a mutation in ABA3 leads to an impairment of freezing tolerance, and not only strengthen the conclusion that ABA is required for full development of freezing tolerance in cold-acclimated plants, but also demonstrate that ABA mediates the constitutive freezing tolerance of Arabidopsis. Gene expression in frs1 mutants was altered in response to dehydration, suggesting that freezing tolerance in Arabidopsis depends on ABA-regulated proteins that allow plants to survive the challenges imposed by subzero temperatures, mainly freeze-induced cellular dehydration. Received: 16 December 1999 / Accepted: 31 March 2000