The Arabidopsis LOS5/ABA3 Locus Encodes a Molybdenum Cofactor Sulfurase and Modulates Cold Stress– and Osmotic Stress–Responsive Gene Expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.     

The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.     

HOS5–a negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana

Osmotic stress activates the expression of many plant genes through ABA-dependent as well as ABA-independent signaling pathways. We report here the characterization of a novel mutant of Arabidopsis thaliana, hos5-1, which exhibits increased expression of the osmotic stress responsive RD29A gene. The expression of several other stress genes are also enhanced by the hos5-1 mutation. The enhanced expression is specific to ABA and osmotic stress because low temperature regulation of these genes is not altered in the mutant. Genetic analysis indicated that hos5-1 is a recessive mutation in a single nuclear gene on chromosome III. Double mutant analysis of hos5-1 and the ABA-deficient aba1-1 as well as the ABA-insensitive abi1-1 mutant indicated that the osmotic stress hypersensitivity of hos5-1 is not affected by ABA deficiency or insensitivity. Furthermore, combined treatments of hos5-1 with ABA and osmotic stress had an additive effect on RD29A-LUC expression. These results suggest that the osmotic stress hypersensitivity in hos5-1 may be ABA-independent. The germination of hos5-1 seeds was more resistant to ABA. However, the hos5-1 mutation did not influence stomatal control and only slightly affected the regulation of growth and proline accumulation by ABA. The hos5-1 mutation reveals a negative regulator of osmotic stress-responsive gene expression shared by ABA-dependent and ABA-independent osmotic stress signaling pathways.     

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.     

Characterization of an Arabidopsis thaliana Mutant that Has a Defect in ABA Accumulation: ABA-Dependent and ABA-Independent Accumulation of Free Amino Acids during Dehydration

In an attempt to elucidate the physiological role of ABA inseed dormancy and the adaptive response to dehydration, we isolatedan ABA-deficient mutant of Arabidopsis thaliana (L.) Heynh.which germinated in the presence of a gibberellin biosyntheticinhibitor. Genetic analysis showed this mutation is a new alleleof a recently reported locus aba2, and therefore has been designatedaba2-2. The levels of endogenous ABA in fresh and dehydratedtissues of the aba2-2 mutant were highly reduced compared tothose of wild-type plants. As a consequence, aba2-2 plants wiltand produce seeds with reduced dormancy. Dark germinated seedlingsof the aba2-2 mutant showed true leaves, which were not observedin those of the wild type, indicating that abal-2 em bryos grewprecociously during seed maturation. In the dehydrated tissuesof the wild-type plants, the levels of free proline, isoleucineand leucine were elevated to a content approximately 100-foldhigher than those in fresh tissues. In contrast to the wild-typeplants, dehydration-induced accumulation of proline was highlysuppressed in the aba2-2 mutant plants while that of leucineand isoleucine accumulated. Furthermore, exogenous applicationof ABA to wild-type plants promoted accumulation of free proline,but not leucine nor isoleucine. These results suggest that dehydration-inducedaccumulation of free leucine and isoleucine is achieved independentof ABA. (Received March 5, 1998; Accepted June 2, 1998)     

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.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

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.     

Time course analysis of ABA and non-ionic osmotic stress-induced changes in water status,chlorophyll fluorescence and osmotic adjustment in Arabidopsis thaliana wild-type (Columbia) and ABA-deficient mutant (aba2)

In the present study, we investigated time course changes of water status including relative water content (RWC), leaf osmotic potential (Ψ_Π), stomatal conductance (g_s), proline (Pro), chlorophyll fluorescence (F_v/F_m) and total chlorophyll content in the Arabidopsis thaliana under PEG-induced drought stress after exogenous ABA treatment. To a better explanation for the role of ABA in the water status of A. thaliana to drought stress, wild-type (Columbia) and ABA-deficient mutant (aba2) of A. thaliana were used in the present study. Moreover, three weeks old Arabidopsis seedlings were applied exogenously with 50 μM ABA and exposed to drought stress induced by 40% PEG8000 (−0.73 MPa) for 6 h, 12 h and 24 h (hours). Our findings indicate that RWC of wild-type and aba2 started to decrease in the first 12 h and 6 h of PEG-induced drought stress, respectively. However, exogenous treatment of 50 μM ABA increased their RWC under drought stress. On the other hand, while Ψ_Π of both genotypes started to decrease in the first 6 h of drought stress, these declines in Ψ_Π were prevented by ABA treatment under stress throughout the experiment; it was more pronounced in aba2 at 24 h. While the highest increase in g_s was obtained in aba2 after 24 h stress, ABA-induced highest decrease in g_s was obtained in the same genotype during 12 h, as compared to PEG-treated group alone. On the other hand, Pro content increased in all treatment groups of ABA-deficient mutant aba2 at 12 h and 24 h. However, Pro content in ABA + PEG treated aba2 plants was higher than in PEG- and ABA-treated plants alone at the end of the 24 h. Drought stress decreased F_v/F_m and total chlorophyll contents of both genotypes while 50 μM ABA alleviated these reductions during drought stress, as compared to PEG stressed plants. On the other hand, 50 μM ABA treatment alone did not create any remarkable effect on F_v/F_m and total chlorophyll contents.These findings indicate that exogenous ABA showed an alleviative effect against damage of drought stress on relative water content, osmotic potential, stomatal conductance, proline, chlorophyll fluorescence and total chlorophyll content of both genotypes during 24 h of drought stress treatment.     

The aba3-1 Mutant of Arabidopsis thaliana Withstands Moderate Doses of Salt Stress by Modulating Leaf Growth and Salicylic Acid Levels

The role of abscisic acid (ABA) and salicylic acid (SA) in salt stress tolerance was studied in Arabidopsis thaliana using mutants that show a defect in hormone biosynthesis or signaling. Plants were subjected to either control conditions (irrigated with nutrient solution) or a moderate salt stress (nutrient solution + 100 mM NaCl), and the response of the aba3, abi4, sid2, and eds5 mutants (with defective ABA or SA biosynthesis/signaling) was compared to that of the wild type (WT). A particular phenotype was observed in the aba3 mutant, which was characterized by reduced plant biomass and lower relative leaf water contents (RWC) under control conditions. However, salt stress reduced growth in the WT, sid2, and eds5 mutants, and to a lesser extent in the abi4 mutant, but not in the aba3 mutant. An analysis of the hormonal balance of leaves revealed that altered SA levels may explain, at least partly, growth changes in the aba3 mutant, under both control and salt stress conditions. The aba3-1 mutant showed higher SA levels than the WT under control conditions and a drastic decrease in the levels of this plant growth regulator under salt stress, an aspect that was not observed in the WT. However, reductions in endogenous SA levels in sid2 and eds5 mutants did not result in increased growth either under control or salt stress conditions. Among the tested genotypes, the aba3 mutant was the only one in which jasmonic acid (JA) levels did not increase in response to salt stress. It is concluded that although ABA deficiency can severely affect plant growth and water relations in aba3 mutants, these plants modulate, among other processes, leaf growth and SA levels, which help them withstand moderate doses of salt stress.     

Scion and Rootstock Effects on ABA-mediated Plant Growth Regulation and Salt Tolerance of Acclimated and Unacclimated Potato Genotypes

Tolerance of salt stress in potato (Solanum tuberosum L.) increased when the plants were pre-exposed to low concentrations of salt (salt acclimation). This acclimation was accompanied by increased levels of abscisic acid (ABA) in the shoot. To further study the role of roots and shoots in this acclimation process, reciprocal grafts were made between a salt-tolerant (9506) and salt-sensitive ABA(−) mutant and its ABA(+) normal sibling potato genotype. The grafted plants were acclimated with 75 or 100 mM NaCl for 3 weeks and then exposed to 150–180 mM NaCl, depending on the salt tolerance of the rootstock. After 2 weeks of exposure to the salt stress, the acclimated and unacclimated plants were compared for physiologic and morphologic parameters. The response to the salt stress was strongly influenced by the rootstock. The salt-tolerant 9506 rootstock increased the salt tolerance of scions of both the ABA-deficient mutant and its ABA(+) sibling. This salt tolerance induced by the rootstock was primarily modulated by salt acclimation and manifested in the scion via increased plant water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential, and is associated with a reduction in leaf necrosis. There was also a pronounced scion effect on the rootstock. Using 9506 as a scion significantly increased root fresh and dry weights, stem diameter, and root water content of ABA(−) mutant rootstocks. Specific evidence was found of the role of exogenous ABA in the enhancement of water status in grafted plants under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and upward water flow in ABA-treated grafted and nongrafted plants and the absence of upward water flow in nontreated grafted plants through NMR imaging. Grafting using either salt-tolerant scions or rootstocks with inherently high ABA levels may positively modify subsequent responses of the plant under salt stress.     

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     

A Novel ABA-Responsive TaSRHP Gene from Wheat Contributes to Enhanced Resistance to Salt Stress in Arabidopsis thaliana

A microarray analysis of the salt-resistant wheat mutant, RH8706-49, revealed a salt-induced gene containing a conserved DUF581 domain. The gene was cloned and designated as Triticum aestivum salt-related hypothetical protein (TaSRHP) and submitted to GenBank (accession no. GQ476575). Over-expression of TaSRHP in wild-type Arabidopsis thaliana cv. Columbia resulted in enhanced resistance to both salt and drought stresses. The sensitivity of the transgenic A. thaliana to abscisic acid (ABA) was also increased compared to that of wild-type plants. Furthermore, transgenic plants accumulated more K⁺ and proline and had a higher osmotic potential and lower Na⁺ content than untransformed plants. Real-time quantitative PCR analysis indicated that expression of TaSRHP was affected by salt, drought, cold, ABA, and other stresses, and expression of other stress-related genes in the transgenic plants differed from those of the control. Results indicate that the wheat TaSRHP gene may enhance the tolerance of plants to multiple abiotic stresses.     

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.     

Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP5CS1 and OsP5CR gene expression during salt stress

Abscisic acid (ABA) applied exogenously at 100 μM prior to and during the salt-stress period induced salt tolerance in both the salt-susceptible (LPT123) and the genetically related salt-resistant (LPT123-TC171) rice lines, enhanced the survival rate by 20%, and triggered proline (Pro) accumulation earlier than that by salt-stress alone, supporting a role for Pro as an osmoprotectant. In both rice lines, salt-stress induced OsP5CS1 gene expression, suggesting that proline accumulation occurs via OsP5CS1 gene expression during salt stress. An increase in the endogenous ABA level was required for the induction of OsP5CS1 gene expression by salt stress. Under salt stress, topical ABA application-induced OsP5CS1 gene expression only in the salt-resistant line but up-regulated OsP5CR gene expression in both rice lines, suggesting that the increased proline accumulation and salt resistance induced by topical ABA application may result from the up-regulation of OsP5CR and not, directly at least, from OsP5CS1. Moreover, exogenous ABA application up-regulates OsCam1-1 (the salt-stress-responsive calmodulin) gene expression, and calmodulin was shown to play a role in the signal transduction cascade in proline accumulation during salt stress. These data suggest the role of the calmodulin signaling cascade and the induction of OsP5CR gene expression in proline accumulation by exogenous ABA application.