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.     

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     

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.     

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 induces the alcohol dehydrogenase gene in Arabidopsis.

Exogenous abscisic acid (ABA) induced the alcohol dehydrogenase gene (Adh) in Arabidopsis roots. Both the G-box-1 element and the GT/GC motifs (anaerobic response element) were required for Adh inducibility. Measurement of endogenous ABA levels during stress treatment showed that ABA levels increased during dehydration treatment but not following exposure to either hypoxia or low temperature. Arabidopsis ABA mutants (aba1 and abi2) displayed reduced Adh mRNA induction levels following either dehydration treatment or exogenous application of ABA. Low-oxygen response was slightly increased in the aba1 mutant but was unchanged in abi2. Low-temperature response was unaffected in both aba1 and abi2 mutants. Our results indicate that, although induction of the Adh gene by ABA, dehydration, and low temperature required the same cis-acting promoter elements, their regulatory pathways were at least partially separated in a combined dehydration/ABA pathway and an ABA-independent low-temperature pathway. These pathways were in turn independent of a third signal transduction pathway leading to low-oxygen response, which did not involve either ABA or the G-box-1 promoter element.     

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 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.     

Genetic evidence that expression of NahG modifies defence pathways independent of salicylic acid biosynthesis in the Arabidopsis-Pseudomonas syringae pv. tomato interaction

The salicylic acid (SA)-induction deficient (sid) mutants of Arabidopsis, eds5 and sid2 accumulate normal amounts of camalexin after inoculation with Pseudomonas syringae pv. tomato (Pst), while transgenic NahG plants expressing an SA hydroxylase that degrades SA have reduced levels of camalexin and exhibit a higher susceptibility to different pathogens compared to the sid mutants. SID2 encodes an isochorismate synthase necessary for the synthesis of SA. NahG was shown to act epistatically to the sid mutant phenotype regarding accumulation of camalexin after inoculation with Pst in eds5NahG and sid2NahG plants. The effect of the pad4 mutation on the sid mutant phenotype was furthermore tested in eds5pad4 and sid2pad4 double mutants, and it was demonstrated that PAD4 acts epistatically to EDS5 and SID2 regarding the production of camalexin after inoculation with Pst. NahG plants and pad4 mutants were also found to produce less ethylene (ET) after infection with Pst in comparison to the wild type (WT) and sid mutants. Both PAD4 and NahG acted epistatically to SID regarding the Pst-dependent production of ET that was found to be necessary for the accumulation of camalexin. Early production of jasmonic acid (JA) 12 h after inoculation with Pst/avrRpt2 was absent in all plants expressing NahG compared to the other mutants tested here. These genetic studies unravel pleiotropic changes in defence signalling of NahG plants that are unlikely to result from their low SA content. This adds unexpected difficulties in the interpretation of earlier findings based solely on NahG plants.     

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.     

Mesophyll conductance decreases in the wild type but not in an ABA‐deficient mutant (aba1) of Nicotiana plumbaginifolia under drought conditions

Under drought conditions, leaf photosynthesis is limited by the supply of CO₂. Drought induces production of abscisic acid (ABA), and ABA decreases stomatal conductance (g_s). Previous papers reported that the drought stress also causes the decrease in mesophyll conductance (g_m). However, the relationships between ABA content and g_m are unclear. We investigated the responses of g_m to the leaf ABA content [(ABA)_L] using an ABA‐deficient mutant, aba1, and the wild type (WT) of Nicotiana plumbaginifolia. We also measured leaf water potential (Ψ_L) because leaf hydraulics may be related to g_m. Under drought conditions, g_m decreased with the increase in (ABA)_L in WT, whereas both (ABA)_L and g_m were unchanged by the drought treatment in aba1. Exogenously applied ABA decreased g_m in both WT and aba1 in a dose‐dependent manner. Ψ_L in WT was decreased by the drought treatment to ?0.7 MPa, whereas Ψ_L in aba1 was around ?0.8 MPa even under the well‐watered conditions and unchanged by the drought treatment. From these results, we conclude that the increase in (ABA)_L is crucial for the decrease in g_m under drought conditions. We discuss possible relationships between the decrease in g_m and changes in the leaf hydraulics.     

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.