Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSITIVE1-dependent pathway

Expression of AtPHO1;H10, a member of the Arabidopsis (Arabidopsis thaliana) PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE1 (COI1). The induction of AtPHO1;H10 expression by wounding and dehydration was dependent on COI1 and was comparable in both the wild type and the OPDA reductase 3-deficient (opr3) mutant. In contrast, induction of AtPHO1;H10 expression by exogenous abscisic acid (ABA) was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA-insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, whereas a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in coregulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis

The formation of lateral roots (LR) is a major post-embryonic developmental event in plants. In Arabidopsis thaliana, LR development is inhibited by high concentrations of NO3(-). Here we present strong evidence that ABA plays an important role in mediating the effects of NO3(-) on LR formation. Firstly, the inhibitory effect of NO3(-) is significantly reduced in three ABA insensitive mutants, abi4-1, abi4-2 and abi5-1, but not in abi1-1, abi2-1 and abi3-1. Secondly, inhibition by NO3(-) is significantly reduced, but not completely abolished, in four ABA synthesis mutants, aba1-1, aba2-3, aba2-4 and aba3-2. These results indicate that there are two regulatory pathways mediating the inhibitory effects of NO3(-) in A. thaliana roots. One pathway is ABA-dependent and involves ABI4 and ABI5, whereas the second pathway is ABA-independent. In addition, ABA also plays a role in mediating the stimulation of LR elongation by local NO3(-) applications.     

Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance

To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.     

The acceptor availability at photosystem I and ABA control nuclear expression of 2-Cys peroxiredoxin-A in Arabidopsis thaliana

The redox-regulated 2-Cys peroxiredoxin-A (2CPA) promoter, which drives expression of a dominant chloroplast antioxidant enzyme, responds to signals originating from the photosynthetic electron transport downstream of PSI. Modulation of CO(2)- and NO(3)(-) -reduction rates in reporter gene plants expressing glucuronidase under control of the Arabidopsis thaliana 2CPA promoter revealed that promoter activity correlates with the availability of electron acceptors at PSI. The photosynthetic redox-regulation can be simulated by oxidant and antioxidant treatments. Inhibitor studies with PD98059 and staurosporine showed that a mitogen-activated protein kinase kinase transmits the oxidative response, while the antioxidant signal is transmitted by a serine/threonine kinase. Analysis of 2CPA promoter regulation in the abscisic acid (ABA)-biosynthetic mutants aba2 and aba3 and the ABA-insensitive mutants abi1 and abi2 support a regulatory circuitry in which the redox signal cross-talks with the ABA-signaling cascade downstream of ABI1 and ABI2.     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation

The mechanisms by which plants respond to reduced water availability (low water potential) include both ABA-dependent and ABA-independent processes. Pro accumulation and osmotic adjustment are two important traits for which the mechanisms of regulation by low water potential, and the involvement of ABA, is not well understood. The ABA-deficient mutant, aba2-1, was used to investigate the regulatory role of ABA in low water potential-induced Pro accumulation and osmotic adjustment in seedlings of Arabidopsis thaliana. Low water potential-induced Pro accumulation required wild-type levels of ABA, as well as a change in ABA sensitivity or ABA-independent events. Osmotic adjustment, in contrast, occurred independently of ABA accumulation in aba2-1. Quantification of low water potential-induced ABA and Pro accumulation in five ABA-insensitive mutants, abi1-1, abi2-1, abi3, abi4, and abi5, revealed that abi4 had increased Pro accumulation at low water potential, but a reduced response to exogenous ABA. Both of these responses were modified by sucrose treatment, indicating that ABI4 has a role in connecting ABA and sugar in regulating Pro accumulation. Of the other abi mutants, only abi1 had reduced Pro accumulation in response to low water potential and ABA application. It was also observed that abi1-1 and abi2-1 had increased ABA accumulation. The involvement of these loci in feedback regulation of ABA accumulation may occur through an effect on ABA catabolism or conjugation. These data provide new information on the function of ABA in seedlings exposed to low water potential and define new roles for three of the well-studied abi loci.     

Abscisic acid negatively regulates post-penetration resistance of Arabidopsis to the biotrophic powdery mildew fungus

Pytohormone abscisic acid(ABA) plays important roles in defense responses.Nonetheless,how ABA regulates plant resistance to biotrophic fungi remains largely unknown.Arabidopsis ABA-deficient mutants,aba2-1 and aba3-1,displayed enhanced resistance to the biotrophic powdery mildew fungus Golovinomyces cichoracearum.Moreover,exogenously administered ABA increased the susceptibility of Arabidopsis to G.cichoracearum.Arabidopsis ABA perception components mutants,abil-1 and abi2-1,also displayed similar phenotypes to ABA-deficient mutants in resistance to G.cichoracearum.However,the resistance to G.cichoracearum is not changed in downstream ABA signaling transduction mutants,abi3-1,abi4-1,and abi5-1.Microscopic examination revealed that hyphal growth and conidiophore production of G.cichoracearum were compromised in the ABA deficient mutants,even though pre-penetration and penetration growth of the fungus were not affected.In addition,salicylic acid(SA) and MPK3 are found to be involved in ABA-regulated resistance to G.cichoracearum.Our work demonstrates that ABA negatively regulates post-penetration resistance of Arabidopsis to powdery mildew fungus G.cichoracearum,probably through antagonizing the function of SA.     

ABA is required for Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLMcol pathway. ABA-deficient and -insensitive mutants in Ler-0 background (abal-3 and abil-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abil-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abil-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. beta-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1co1)pad3 and rlmlLerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1col. ABII, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1co, expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABIJ activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.     

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

 The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (β-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUSArabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression. Received: 23 May 1999 / Accepted: 3 December 1999     

ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity

In most plant species, a decrease in atmospheric humidity at the leaf surface triggers a decrease in stomatal conductance. While guard cells appear to respond to humidity‐induced changes in transpiration rate, as opposed to relative humidity or vapour pressure difference, the underlying cellular mechanisms for this response remain unknown. In the present set of experiments, abscisic acid (ABA)‐deficient (aba1) and ABA‐insensitive (abi1‐1 and abi2‐1) mutants of Arabidopsis thaliana were used to test the hypothesis that the humidity signal is transduced by changes in the flux or concentration of ABA delivered to the stomatal complex in the transpiration stream. In gas exchange experiments, stomatal conductance was as sensitive to changes in vapour pressure difference in aba1, abi1‐1 and abi2‐1 mutant plants as in wild‐type plants. These experiments appear to rule out an obligate role for either the concentration or flux of ABA or ABA conjugates as mediators of the guard cell response to atmospheric water potential. The results stand in contrast to the well‐established role of ABA in mediating guard cell responses to decreases in soil water potential.