JXB Advance Access originally published online on December 9, 2005
Journal of Experimental Botany 2006 57(1):201-212; doi:10.1093/jxb/erj026
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RESEARCH PAPER |
Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation
Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA
* To whom correspondence should be addressed. E-mail: paul.verslues{at}ucr.edu
Received 17 June 2005; Accepted 24 October 2005
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Abstract |
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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.
Key words: ABA (abscisic acid), ABA sensitivity, abi mutants, abiotic stress, Arabidopsis thaliana, low water potential, osmotic adjustment, proline
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Introduction |
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Low water potential (
w) and subsequent changes in turgor and water content elicit many responses that allow plants to adapt to stress and, to a certain extent, continue to grow and develop. The large and rapid accumulation of ABA occurring in response to low w is a critical component in the activation of downstream responses at both the physiological level, such as stomatal regulation (Assmann, 2003
), and the molecular level through numerous changes in gene expression (Bray, 1993, 2003; Shinozaki et al., 2003; Zhu, 2002). ABA-dependent responses interact with ABA-independent events and changes in sensitivity to ABA to generate a co-ordinated low w response.
In general, two key sets of observations indicate that one must look beyond ABA accumulation itself to understand the low w response. First, some low w responses do not require wild-type levels of ABA. For example, analysis of several Arabidopsis thaliana mutants with reduced ABA accumulation at low w has shown that a number of low w-induced genes are induced to wild-type or near wild-type levels despite the reduced level of ABA (Shinozaki et al., 2003). Also, Assmann et al. (2000) reported that the Arabidopsis ABA-deficient mutant aba1, as well as the ABA-insensitive mutants abi1 and abi2, was unaffected in stomatal closure in response to reduced humidity (although all of these mutants are impaired in low w-induced stomatal closure). Second, ABA application to unstressed plants does not always simulate the effects of low w. This has been demonstrated for the expression of the stress- and ABA-regulated gene le25 (Imai et al., 1995) and ABA-mediated root growth maintenance and shoot growth repression in response to low w (Sharp and LeNoble, 2002; Sharp et al., 1994). Whether or not these differences are the result of sensing and signalling events that do not involve ABA or are the result of altered response to ABA under low w is unclear. For many low w responses, such as Pro accumulation and osmotic adjustment, little or no data are available that would allow the relative importance of ABA accumulation, changes in ABA sensitivity, and ABA-independent regulation to be assessed.
In Arabidopsis, a series of ABA-insensitive (abi) mutants have been isolated. The abi mutants all exhibit ABA-insensitive seed germination, but differ in their effects on other ABA-regulated responses (Finkelstein et al., 2002). ABI1 and ABI2 are type 2C protein phosphatases that act as negative regulators of ABA signalling (Leung et al., 1997). ABI1 and ABI2 are expressed at a higher level in vegetative tissues than in seeds and are induced by ABA and osmotic stress (Leung et al., 1997). ABI3 encodes a B3 domain transcription factor (Giraudat et al., 1992) and is expressed mainly in seeds and meristematic tissue with a low level of expression in vegetative tissue (Finkelstein et al., 2002). ABI4 encodes an APETALA2 domain transcription factor (Finkelstein et al., 1998) and ABI5 encodes a bZIP domain transcription factor (Finkelstein and Lynch, 2000). ABI4 and ABI5 are expressed most abundantly in developing seeds, but both also have low levels of vegetative expression (Finkelstein et al., 1998; Finkelstein and Lynch, 2000). Despite the importance of understanding the signalling events regulating plant stress responses, the low w responses of abi1 through abi5, have not been fully characterized. abi1-1, abi2-1, and abi3 have all been reported to have altered responses to exogenous ABA, in addition to effects on seed germination (Finkelstein, 1994; Finkelstein and Somerville, 1990; Suzuki et al., 2001), raising the possibility that the abi mutants could affect ABA-regulated low w responses as well. However, Cramer (2002) found no difference in dry weight, ABA, and ion content between abi1-1, abi2-1, abi3, and wild type exposed to high salinity.
Pro accumulation is an important low w response; although its function and regulation are not well understood. Pro is a compatible solute that can have a major role in osmotic adjustment (Voetberg and Sharp, 1991) and may also have a number of other protective roles. These include protecting protein and membrane structure (Chen and Murata, 2002; Yancey et al., 1982), scavenging reactive oxygen (Hong et al., 2000; Smirnoff and Cumbes, 1989), and eliminating excess reductant or regulating cellular redox status (Hare et al., 1998). In addition, Pro, or its catabolic intermediate 
1-pyrroline-5-carboxylate (P5C) has been proposed to have a role in the metabolic signalling of carbohydrate status (Hellmann et al., 2000).
There is evidence that ABA is required for low w-induced Pro accumulation. Blocking ABA accumulation using either the ABA-deficient maize mutant vp5 or fluridone dramatically reduced Pro deposition in low w-treated primary roots (Ober and Sharp, 1994). In agreement with this, Strizhov et al. (1997) found that salt stress induction of the gene encoding the rate-limiting enzyme in stress-induced Pro biosynthesis, P5C SYNTHASE1 (P5CS1), was inhibited in the ABA-deficient mutant aba1 and in abi1. On the other hand, Savoure et al. (1997) have stated that expression of P5CS1 was induced independently of ABA in response to salinity or low w. Thus, the possible regulatory mechanisms controlling low w-induced Pro accumulation include ABA-dependent and independent factors, as well as changes in ABA sensitivity.
The ABA-deficient mutant, aba2-1, was used to investigate the role of ABA in Pro accumulation and osmotic adjustment. It was observed that Pro accumulation requires ABA accumulation, yet ABA alone is not sufficient to induce the Pro content observed at low w. Osmotic adjustment, by contrast, did not require wild-type ABA levels. To understand the mechanisms of ABA-dependent regulation of Pro accumulation further, low w-induced ABA and Pro accumulation in abi1-1, abi2-1, abi3, abi4, and abi5 were assayed and new phenotypes were found for several of these mutants. abi1-1 and abi2-1 have increased ABA accumulation indicating a role for these loci in controlling feedback regulation of ABA accumulation. abi4 alters Pro accumulation in a sucrose-dependent manner indicating a role for ABI4 in connecting ABA and sugar signalling in the low w response.
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Materials and methods |
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Plant material
Seed stocks of the abi mutants were obtained from the Arabidopsis Biological Resource Center and the ABA-insensitive germination phenotype of each line was verified (Assmann et al., 2000
). aba2-1 was obtained from the same source and its genotype verified by its reduced growth and ABA accumulation at low w. Data for each mutant was compared with that of the appropriate wild-type ecotype. Some experiments were performed with the Bensheim ecotype which has similar levels of Pro and ABA accumulation as Col (Verslues and Bray, 2004).
Low w and ABA treatments
Low w and ABA treatments were performed using vertically-positioned agar plates as previously described (van der Weele et al., 2000; Verslues and Bray, 2004). Seeds were plated on basal media (half-strength MS media with 2 mM MES buffer, pH 5.7, solidified with agar). Media were prepared without the addition of sucrose or other sugars unless otherwise noted. Prior to seed plating, the plates were overlain with a nylon mesh to facilitate transfer of seedlings to a new plate. After stratification for 3 d at 4 °C, plates were transferred to a growth room (23 °C, 16 h light period, 150–180 µE cm–2 min–1 light intensity) and placed vertically inside a humidified plexiglass chamber to prevent the plates from drying and to allow the seedlings to grow along the surface of the agar. After 3 or 4 d of growth, seedlings were transferred using the nylon mesh to low w, ABA-containing, or basal media agar plates. For low w treatment, PEG-infused agar plates were prepared by overlaying plates (100x20 cm) containing 20 ml of basal media with 30 ml of PEG-8000 solution. After overnight equilibration, excess PEG solution was removed. PEG concentrations of 250, 400, 550, and 700 g l–1 were used to produce plates with a final w of –0.5, –0.7, –1.2, and –1.7 MPa, respectively. Agar w was verified using a vapour pressure osmometer (Model 5100C, Wescor, Logan, UT). ABA treatments were performed by adding S(+)-ABA (Lomon BioTechnology, Sichuan, China), dissolved in a small volume of ethanol, to the basal media after autoclaving. Controls with ethanol only added to the basal media showed no effect on seedling Pro or ABA content. Combined low w and ABA treatments were performed by adding the indicated concentration of ABA to both the basal media before the addition of PEG and to the PEG solution overlaid on the agar media.
Quantification of Pro, ABA and osmotic adjustment
Pro was assayed on water-extracted seedlings using the ninhydrin assay of Bates et al. (1973). Samples consisted of 10–30 seedlings depending on the experimental treatment. ABA was assayed by radioimmunoassay (Bray and Beachy, 1985). Seedling samples for ABA analysis consisted of 20–100 seedlings (15–200 mg of tissue) depending on the experimental treatment. For ABA quantification, seedlings were removed from the agar plate using the nylon mesh and twice rinsed (each rinse <10 s) with a NaCl solution of the same w as the agar to remove any PEG or exogenous ABA that may interfere with the assay of seedling internal ABA. Seedlings were then blotted and weighed.
Measurements of relative water content (RWC) and osmotic potential (s), and calculation of osmotic adjustment were performed as described previously (Verslues and Bray, 2004). Seedling solute content was quantified by homogenizing whole seedlings and measuring s of the cell sap using a vapour pressure osmometer. Seedling RWC was measured by removing seedlings from the agar plate using the nylon mesh, briefly blotting to remove excess liquid and weighing. Seedlings were then incubated in ice-cold water for 2–3 h to rehydrate, blotted, reweighed, and dried overnight at 65 °C. Dry weight was recorded, the weight of the nylon mesh subtracted from all measurements, and RWC calculated as: (fresh weight–dry weight)/(hydrated weight–dry weight)x100. s at 100% RWC (s100) was calculated by multiplying the RWC by the s.
Data reported for these assays represent the combined mean of at least two independent experiments with two or three samples collected in each experiment. Statistically significant differences were determined by standard two-tailed t-test with P-values as noted in the text or figure legends.
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Results |
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ABA accumulation is required to induce Pro accumulation at low
wPro and ABA both accumulate in 3-d-old Arabidopsis seedlings when exposed to a constant, reproducible low
w stress using a PEG-infused agar plate system (van der Weele et al., 2000; Verslues and Bray, 2004); although the pattern of accumulation of these two metabolites differs over a 96-h experimental time-course (Fig. 1). To begin to determine the relationship between low w-induced Pro and ABA accumulation, Pro and ABA contents of the ABA-deficient mutant, aba2-1, were compared with wild type after the transfer of seedlings to either –0.25 MPa control plates or –1.2 MPa PEG-infused plates (Fig. 1). Pro accumulation did not differ between wild-type and aba2-1 seedlings in the first 8 h of the stress treatment. However, after 8 h there was no further increase in Pro content in the ABA-deficient mutant. 96 h after transfer to low w, the Pro content of the wild type was 35-fold greater than the control, while that of aba2-1 was only 5-fold greater. ABA accumulated rapidly in wild-type seedlings on –1.2 MPa plates and reached a maximum at approximately 8 h (Fig. 1B). ABA levels then declined to a steady-state level that was 15-fold higher than the unstressed level. The decline in ABA content after 24 h is consistent with a number of other reports of rapid ABA accumulation followed by a decline with longer exposure to stress (Cowan et al., 1997). aba2-1 had less than a 2-fold increase in ABA content in response to the 96 h low w treatment and lacked the initial peak in ABA content observed at 8 h in the wild type (Fig. 1B).
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0.05.These results indicate that, despite the different temporal pattern of ABA and Pro accumulation, ABA accumulation was required for the high level of low
w-induced Pro accumulation of the wild type. In this experimental system, the partial Pro accumulation that occurred in the first 8 h of low w treatment was not dependent on an increase in seedling ABA content, while the Pro accumulation occurring in the following 72–96 h required the elevated ABA content of the wild type. A portion of the Pro accumulation could be attributed to a decrease in seedling water content after transfer to low w. Seedling water content decreased as much as 40% in the first 8 h after transfer to –1.2 MPa and recovered to 15–20% below the unstressed level by 96 h (data not shown; Verslues and Bray, 2004). However, the level of Pro accumulation that occurred was larger than could be explained by decreased water content alone. Confirmation that ABA deficiency caused the reduced Pro accumulation in aba2-1 was obtained by applying exogenous ABA at –1.2 MPA to restore the ABA content of aba2-1 to the wild-type level. For seedlings exposed to –1.2 MPa the Pro content of aba2-1 in the presence of 0.5 µM S(+)-ABA was not significantly different from the wild type exposed only to the –1.2 MPa treatment (Fig. 2; an asterisk indicates a significant difference from the wild type at 0 µM ABA). Thus, the decreased Pro accumulation of aba2-1 was complemented by the addition of ABA, indicating that ABA is required for Pro accumulation in this system.
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Exogenous ABA at high
w does not reproduce the low w induction of Pro accumulationReduced Pro accumulation in the ABA-deficient mutant, aba2-1, demonstrated that ABA accumulation was necessary to achieve a high steady-state level of Pro after a 96 h low
w treatment. Yet, the question remains whether ABA is sufficient to induce the high level of Pro accumulation that occurs at low w. To address this question, Pro and ABA contents were measured after seedlings were transferred to plates of a range of ABA concentrations at high w or to PEG-infused plates of a range of w.
ABA treatment of seedlings at high w (–0.25 MPa) caused a small (3.5-fold) increase in seedling Pro content despite a large increase in the internal ABA content of the seedlings (Fig. 3A). When these data were plotted as Pro content versus log internal seedling ABA content (Fig. 3B), there was a linear (r2=0.95) relationship. It has previously been observed that application of concentrations as high as 100 µM ABA have little additional effect on Pro accumulation (Verslues and Bray, 2004). Thus, the range of ABA treatments examined here are within the range [the linear portion of a sigmoidal dose–response curve (Weyers et al., 1995)] where change in exogenous ABA concentration should have the greatest effect on seedling Pro content. These data suggest that in unstressed seedlings the sensitivity to ABA with respect to Pro accumulation is low.
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By contrast, Pro and ABA content increased proportionally to decreasing
w over the range of –0.25 MPa to –1.7 MPa (Fig. 3C). The plot of seedling Pro content versus log internal seedling ABA content at low w (Fig. 3D) is consistent with an increased sensitivity to ABA in low w-treated seedlings with respect to Pro accumulation. The response to ABA at low water potential could be explained by factors that act downstream of ABA and amplify the ABA signal; alternatively, ABA-independent factors that act directly to stimulate Pro accumulation at low w without interacting with ABA may also exist.
ABA accumulation is not required for osmotic adjustment
Osmotic adjustment is an accumulation of solutes in response to low w that decreases cellular osmotic potential (s) and functions to prevent cellular water loss (Zhang et al., 1999). Osmotic adjustment in wild-type and aba2-1 seedlings was quantified to determine the involvement of ABA in osmotic adjustment. The fact that transpiration is minimal in this experimental system is especially advantageous in that osmotic adjustment can be quantified in response to a constant severity of low w without the confounding effects of increased transpirational water loss in the aba2-1 mutant. Seedling RWC and s were measured over a range of agar w and these values were used to calculate s100 (Babu et al., 1999; Verslues and Bray, 2004). The RWC (Fig. 4A) and s (Fig. 4B) of aba2-1 and wild type was similar in seedlings exposed to a range of agar w. Thus, s100 did not differ between the mutant and wild type (Fig. 4C). This was verified by fitting regression lines to the s100 data for each genotype. Statistical comparison of the regression lines (by F-test) revealed no significant difference between the mutant and wild type and a single line was fitted to the data of both genotypes (s100=0.30 w= –0.43; r2=0.92). The level of osmotic adjustment (131 mM MPa–1) was calculated from the slope of the line and was similar to previous measurements for the ecotype Bensheim (150 mM MPa–1; Verslues and Bray, 2004). Thus, in aba2-1, wild-type levels of osmotic adjustment can occur without wild-type levels of ABA accumulation.
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abi1-1, abi2-1 and abi4 have altered ABA or Pro accumulation in response to low
w or exogenous ABAAnalysis of Pro and ABA contents of wild type indicated a role of ABA signalling in low
w-induced Pro accumulation, thus Pro and ABA accumulation were assayed in five ABA-insensitive (abi) mutants to determine if these mutants are altered in Pro accumulation. Since the five abi mutants are in three different Arabidopsis ecotypes (Ler, Col, and WS) and the different ecotypes have different Pro contents, each mutant was compared to its wild type. Ler had the highest Pro content at both –0.25 and –1.2 MPa followed by WS and Col (P <0.04; Fig. 5A). WS had significantly higher ABA content than Ler and Col (P <0.04) at both –0.25 and –1.2 MPa (Fig. 5B). Only one of the abi mutants, abi1-1, had a significant reduction in low w-induced Pro accumulation (Fig. 5A); although, the 28% reduction was not as great as the 80% reduction of the ABA-deficient mutant, aba2-1. abi1-1 seedlings also accumulated less Pro than wild type in response to 2 µM S(+)-ABA treatment (Fig. 5C). Surprisingly, abi1-1 seedlings had 2–4-fold higher ABA content than Ler in response to both low w (Fig. 5B) and exogenous ABA at high w (Fig. 5D). abi2-1 seedlings also had higher ABA contents. Both abi1-1 and abi2-1 have an approximately 4–5-fold reduction in Pro content per unit of ABA content at low w compared with the wild type (Table 1).
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Of the other ABA-insensitive mutants, only abi4 had significantly lower Pro content (31% lower) than its wild type in response to exogenous ABA at high
w. However, abi4 had greater Pro content than the wild type after the low w treatment (Fig. 5A). The ABA content of abi4 did not differ from the wild type (Fig. 5B, D) resulting in a greater Pro content per unit of ABA at low w than the wild type (Table 1). No effect on Pro or ABA accumulation was seen in abi3 and abi5 (Fig. 5) relative to their parental ecotypes. This is consistent with the conclusions of Brady et al. (2003) who placed abi3 and abi5 on a different branch of ABA signalling pathways than abi4 and downstream of abi1-1 and abi2-1.
abi1-1 and abi2-1 have altered ABA homeostasis
The increased ABA accumulation in the abi1-1 and abi2-1 mutants indicates that ABI1 and ABI2 are regulators of ABA content as well as ABA responses. A time-course of ABA accumulation in abi1-1 and abi2-1 after transfer to low w and the response of abi1-1 and abi2-1 to combined ABA and low w treatments was also quantified. In both abi1-1 and abi2-1, the peak ABA content that occurred approximately 8 h after transfer to low w was 1.5–2-fold higher than the wild type Ler (Fig. 6A). ABA content in the mutants then declined, following a similar pattern as Ler, but remained higher than the wild type at the end of the 96 h time-course.
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Treatment of Ler seedlings with 0.5 or 2 µM ABA at –1.2 MPa increased seedling ABA content up to 2-fold, whereas abi1-1 and abi2-1 seedlings had an approximately 4-fold increase (Fig. 6B). This is similar to the 2–4-fold greater ABA content of abi1-1 and abi2-1 when 2.0 µM exogenous ABA was applied to unstressed seedlings (Fig. 5D). The increased ABA content of abi1-1 and abi2-1 was not dependent on the source of ABA; both exogenous ABA and ABA synthesized in response to low
w accumulated to a greater extent in abi1-1 and abi2-1 than the wild type. The increased ABA accumulation of abi1-1 and abi2-1 was also not affected by low w; ABA applied at either high or low w caused a similar increase in the ABA content of abi1-1 and abi2-1 relative to the wild type. The simplest explanation for these data is that abi1 and abi2 have a decreased rate of ABA turnover, caused by a decrease in ABA catabolism and/or conjugation. However, these experiments cannot directly determine the metabolic mechanisms involved in the increased ABA content of the abi1-1 and abi2-1 mutants.
ABI4 is involved in the interaction of ABA, sugar and low w in regulating Pro accumulation
ABA has been implicated in the ability of Arabidopsis seedlings to respond to signals generated by sugars and the abi4 mutant is insensitive to sugar with respect to seedling development (Laby et al., 2000; Arenas-Huertero et al., 2000; Rook et al., 2001). If sugars also influence Pro metabolism, as has been previously proposed (Hellmann et al., 2000), this may provide an explanation for the altered Pro accumulation of abi4. Pro contents of abi4 seedlings and its wild type, Col, were quantified after treatment with a range of sucrose contents applied at low w (Fig. 7A). Seedlings were germinated and grown on sucrose-free media prior to transfer to –1.2 MPa PEG-infused plates containing sucrose. The addition of sucrose increased low w-induced Pro accumulation for both genotypes and the Pro content of abi4 was greater than wild type at all sucrose treatments except at 3% sucrose, the highest concentration tested. Because abi4 had a higher Pro content than the wild type in the absence of sucrose, the relative stimulation of Pro accumulation by sucrose was greater in the wild type than in abi4.
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In the absence of low
w, the sucrose treatment did not affect steady-state Pro accumulation in either Col or abi4 (Fig. 7B). As reported in Fig. 5B, exogenous ABA promoted Pro accumulation and this response was partially blocked in abi4. Addition of 0.5% sucrose to the media blocked ABA-induced Pro accumulation in the wild type and, to a lesser extent, in abi4 (Fig. 7B). The combined results indicate that the wild type ABI4 gene product is a negative regulator of low w-induced Pro accumulation whose effect is decreased by high levels of sugar. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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ABA is known to be an important regulator of low
w responses, but not all ABA-dependent responses utilize the same signalling intermediates or are affected in the same manner by changes in ABA content (Zhu, 2002). The importance of ABA-dependent and ABA-independent signalling pathways varies among different low w responses. Evidence is presented here that ABA accumulation and changes in ABA sensitivity are important for the regulation of Pro accumulation, but not osmotic adjustment. The ABA-insensitive mutants, abi1-1, abi2-1, and abi4, have altered ABA and Pro accumulation. This extends the range of phenotypes known to be regulated by these important ABA-signalling loci. A simplified model integrating these results is presented in Fig. 8.
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ABA accumulation and response
The regulation of ABA content is complex, involving ABA synthesis as well as ABA catabolism and conjugation (Zeevaart, 1999
). Under stress, it has been observed that ABA content is correlated with the degree to which the stress alters plant water status as measured by changes in turgor and RWC (Pierce and Raschke, 1980; Verslues and Bray, 2004; Zhang and Davies, 1987). Thus, multiple aspects of ABA metabolism may be involved in a homeostasis mechanism preventing excess ABA accumulation and matching ABA content to the type and severity of the stress to which the plant is exposed. The increased ABA accumulation of abi1-1 and abi2-1 indicates that the ABI1 and ABI2 protein phosphatase 2Cs are involved in ABA homeostasis. The most straightforward explanation accounting for the increased ABA content of abi1-1 and abi2-1 in response to both low w and exogenous ABA is that these loci are involved in a feedback mechanism that regulates the turnover of ABA (Fig. 8). However, additional experiments that directly examine the rate of ABA turnover in abi1 and abi2 will be needed to determine which step of ABA metabolism is affected by ABI1 and ABI2. This uncertainty is indicated in Fig. 8 by the dashed line connecting ABI1 and ABI2 feedback regulation to ABA degradation/conjugation. Since abi1-1 and abi2-1 are both dominant alleles and recessive alleles of these genes are hypersensitive to ABA (Merlot et al., 2001), it can be hypothesized that recessive alleles of ABI1 and ABI2 would have a reduced level of low w-induced ABA accumulation indicating that ABI1 and ABI2 are negative regulators of ABA catabolism or conjugation (or positive regulators of ABA synthesis).
Further understanding of feedback regulation by ABA and the rates of ABA catabolism and turnover, as well as the possible role of ABI1 and ABI2 in controlling these processes, are necessary to understand the regulation of ABA accumulation by low w. Half-life values of exogenously applied ABA range from less than 1 h in the maize root apex (Ribaut et al., 1996) to 3–10 h in cell cultures and leaves (Balsevich et al., 1994; Zeevaart and Creelman, 1988). A limited number of studies comparing ABA catabolism in turgid and wilted leaves have either found no difference (Cornish and Zeevaart, 1984; Murphy, 1984) or decreased ABA catabolism in stressed leaves (Cowan and Railton, 1987). Impaired ABA degradation leading to increased ABA accumulation has previously been observed in pew1, a phytochrome-chromophore-deficient mutant of tomato (Kraepiel et al., 1994). Several investigators have suggested that ABA accumulation, application of exogenous ABA, or transgenic approaches to increase ABA synthesis stimulate ABA catabolism (Cutler and Krochko, 1999; Qin and Zeevaart, 2002; Xiong and Zhu, 2003; Zeevaart, 1999).
Interaction of low w, Pro, ABA, and sugar
The role of ABA in regulating Pro accumulation has been a matter of some confusion and debate (Hare et al., 1998, 1999; Ober and Sharp, 1994; Savoure et al., 1997; Strizhov et al., 1997). The results presented here demonstrate that while ABA accumulation is required for Pro accumulation, ABA accumulation alone is not sufficient to elicit the levels of Pro accumulation observed at low w. For both wild-type and aba2-1 seedlings, application of 2.0 µM ABA at –1.2 MPa increased seedling ABA content by more than 2-fold compared with wild-type seedlings subjected to –1.2 MPa alone (Fig. 2B), however, this exogenous ABA treatment did not significantly increase the Pro content (Fig. 2A). This may indicate the existence of additional mechanisms of Pro homeostasis that maintain seedling Pro content at a level appropriate to the stress severity, even if ABA content is increased to a higher than normal level. Alternatively, the perception or response to ABA at –1.2 MPa may already be saturated by the wild-type level of ABA. However, more severe stress treatments (–1.7 MPa, Fig. 3C) did lead to increased accumulation of both ABA and Pro. Thus, both of these regulatory factors, severity of low w stress and ABA, must increase in tandem to increase Pro accumulation further.
Sugars also affect Pro accumulation. This is perhaps not surprising; a major change in metabolism such as increased Pro synthesis would probably be responsive to the levels of substrate and reductant needed for Pro synthesis. Several studies have found that sugars can stimulate Pro accumulation (Pesci, 1993; Stewart et al., 1966). However, recent data indicate a more complex interaction. Hellmann et al. (2000) found that the reduced sugar response1 (rsr1) mutant, which was originally isolated as being impaired in sugar sensing, is also hypersensitive to exogenous Pro. The Pro hypersensitivity of rsr1 can be alleviated by the addition of glucose or sucrose to the media (Hellmann et al., 2000). This indicates that Pro metabolism may respond directly to sugar-sensing mechanisms. In addition, several mutants originally identified as ABA-deficient (aba1, aba2, and aba3) or ABA-insensitive (abi4 and abi5) are also sugar-insensitive (Arenas-Huertero et al., 2000; Laby et al., 2000; Rook et al., 2001). The increased Pro accumulation observed in abi4 in this study indicates that ABI4 may be part of a mechanism to restrict Pro accumulation when carbohydrate status is low (Fig. 8).
Although sugar can influence the level of Pro accumulation, sugar cannot substitute for ABA in inducing Pro accumulation at low w. aba2-1, a sugar-insensitive mutant, accumulates very low levels of Pro at low w because ABA accumulation is required for Pro accumulation. High levels of Pro accumulation can only be induced by the interaction of several signals including ABA, sugar, and osmoregulatory signals (Fig. 8; Verslues and Bray, 2004).
Osmotic adjustment does not require wild-type levels of ABA accumulation
Analysis of aba2-1 demonstrated that seedling osmotic adjustment was not dependent on ABA accumulation. This agrees with a previous characterization of the lwr2 mutant which has reduced osmotic adjustment, but is unaffected in ABA-responsive Pro accumulation or transpirational water loss (Verslues and Bray, 2004). Both the lwr2 and aba2-1 data demonstrate that osmoregulatory control of cellular solute and water content is distinct from the stomatal regulation of water loss and is controlled by different signalling mechanisms that do not require wild-type levels of ABA. Assaying osmotic adjustment under conditions of constant low w stress and minimal transpiration was essential to quantify the osmotic adjustment phenotype of aba2-1 accurately and to separate osmoregulatory responses from stomatal-regulated water loss.
LaRosa et al. (1987) did observe increased osmotic adjustment in ABA-treated tobacco suspension cells exposed to salt stress. Most of the increase in solute content was caused by reducing sugars and Pro. One possible explanation is that ABA-stimulated accumulation of these compounds may prevent damage by NaCl toxicity and thus have a protective effect on metabolism which, in turn, allows further solute accumulation to occur. Whether or not ABA application can stimulate osmotic adjustment induced by non-ionic low w, where NaCl toxicity is not a factor, is not known. LaRosa et al. (1987) also observed a strong correlation between Pro content and cellular s, suggesting an osmoregulatory component in controlling Pro accumulation.
The observation that wild-type levels of osmotic adjustment could occur despite the reduced Pro accumulation of aba2-1 raises the question of whether Pro accumulation is an essential component of osmotic adjustment. Pro is largely contained in the relatively small volume of the cytoplasm (Hare and Cress, 1997; Hare et al., 1998; Leigh et al., 1981) and within the cytoplasm can reach concentrations that are significant in osmotic adjustment. This has been directly demonstrated in studies of Pro accumulation in the unvacuolated cells of the maize root tip where Pro concentrations can reach as high as 120 mM (Ober and Sharp, 1994; Verslues and Sharp, 1999; Voetberg and Sharp, 1991). Thus, an inhibition of Pro accumulation, such as occurs in aba2-1, may not have a large impact of the overall solute content of highly vacuolated tissues, but can have a large impact on the cytoplasmic solute content. In the case of aba2-1, it is hypothesized that Pro normally accumulated in the cytoplasm as part of osmotic adjustment is replaced by other solutes such as K+. These solutes may be less effective than Pro at protecting cytoplasmic structure and function. Thus, promoting the accumulation of protective solutes in the cytoplasm, rather than simply promoting greater total solute accumulation, may be a function of ABA that increases cellular tolerance to low w and other stresses.
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This work was supported by the National Science Foundation (grant no. GE-9355042 to EAB). PEV was also supported by the Graduate Division and the Department of Botany and Plant Sciences, University of California, Riverside. We thank Mayuki Tanaka and Ramon Barajas for assistance in the laboratory and Drs Linda Walling and Patricia Springer (Department of Botany and Plant Sciences, University of California, Riverside) for useful discussion.
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Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA. 
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|---|
Arenas-Huertero F, Arroyo A, Zhou L, Sheen J, Leon P. 2000. Analysis of Arabidopsis glucose-insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes and Development 14, 2085–2096.
Assmann SM. 2003. OPEN STOMATA1 opens the door to ABA signalling in Arabidopsis guard cells. Trends in Plant Science 5, 151–153.
Assmann SM, Snyder JA, Lee YRJ. 2000. ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity. Plant, Cell and Environment 23, 387–395.[CrossRef]
Babu RC, Pathan MS, Blum A, Nguyen HT. 1999. Comparison of measurement methods of osmotic adjustment in rice cultivars. Crop Science 39, 150–158.
Balsevich JJ, Cutler AJ, Lamb N, Friesen LJ, Kurz EU, Perras MR, Abrams SR. 1994. Response of cultured maize cells to (+)-abscisic acid, (–)-abscisic acid, and their metabolites. Plant Physiology 106, 135–142.[Abstract]
Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline in water-stress studies. Plant and Soil 39, 205–207.[CrossRef][Web of Science]
Brady SM, Sarkar SF, Bonetta D, McCourt P. 2003. The ABSCISIC ACID INSENSITIVE3 (ABI3) gene is modulated by farnesylation and is involved in auxin signalling and lateral root development in Arabidopsis. The Plant Journal 34, 67–75.[CrossRef][Web of Science][Medline]
Bray EA. 1993. Molecular responses to water deficit. Plant Physiology 103, 1035–1040.[Web of Science][Medline]
Bray EA. 2003. Abscisic acid regulation of gene expression during water-deficit stress in the era of the Arabidopsis genome. Plant, Cell and Environment 25, 153–161.[CrossRef]
Bray EA, Beachy RN. 1985. Regulation by ABA of ß-conglycinin expression in cultured developing soybean cotyledons. Plant Physiology 79, 746–750.
Chen THH, Murata N. 2002. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Current Opinion in Plant Biology 5, 250–257.[CrossRef][Web of Science][Medline]
Cornish K, Zeevaart JAD. 1984. Abscisic acid metabolism in relation to water stress and leaf age in Xanthium strumarium. Plant Physiology 76, 1029–1035.
Cowan AK, Railton ID. 1987. The catabolism of (±)-abscisic acid by excised leaves of Hordeum vulgare L. cv. Dyan and its modification by chemical and environmental factors. Plant Physiology 84, 157–163.
Cowan AK, Richardson GR, Maurel JCG. 1997. Stress-induced abscisic acid transients and stimulus-response-coupling. Physiologia Plantarum 100, 491–499.[CrossRef]
Cramer GR. 2002. Response of abscisic acid mutants of Arabidopsis to salinity. Functional Plant Biology 29, 561–567.[CrossRef]
Cutler AJ, Krochko JE. 1999. Formation and breakdown of ABA. Trends in Plant Science 4, 472–478.[CrossRef][Web of Science][Medline]
Finkelstein RR. 1994. Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations. The Plant Journal 5, 765–771.[CrossRef][Web of Science]
Finkelstein RR, Gampala SSL, Rock CD. 2002. Abscisic acid signalling in seeds and seedlings. The Plant Cell 14, S15–S45.
Finkelstein RR, Lynch TJ. 2000. The Arabidopsis abscisic acid response gene abi5 encodes a basic leucine zipper transcription factor. The Plant Cell 12, 599–609.
Finkelstein RR, Somerville CR. 1990. Three classes of abscisic acid (ABA)-insensitive mutations of Arabidopsis define genes that control overlapping subsets of ABA responses. Plant Physiology 94, 1172–1179.
Finkelstein RR, Wang ML, Lynch TJ, Rao S, Goodman HM. 1998. The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA2 domain protein. The Plant Cell 10, 1043–1054.
Giraudat J, Hauge B, Valon C, Smalle J, Parcy F, Goodman H. 1992. Isolation of the Arabidopsis ABI3 gene by positional cloning. The Plant Cell 4, 1251–1261.
Hare PD, Cress WA. 1997. Metabolic implication of stress-induced proline accumulation in plants. Plant Growth Regulation 21, 79–102.[CrossRef][Web of Science]
Hare PD, Cress WA, Van Staden J. 1998. Dissecting the roles of osmolyte accumulation during stress. Plant, Cell and Environment 21, 535–553.[CrossRef]
Hare PD, Cress WA, van Staden J. 1999. Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. Journal of Experimental Botany 50, 413–434.
Hellmann H, Funck D, Rentsch D, Frommer WB. 2000. Hypersensitivity of an Arabidopsis sugar signalling mutant toward exogenous proline application. Plant Physiology 123, 779–790.
Hong ZL, Lakkineni K, Zhang ZM, Verma DPS. 2000. Removal of feedback inhibition of 1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiology 122, 1129–1136.
Imai R, Moses MS, Bray EA. 1995. Expression of an ABA-induced gene of tomato in transgenic tobacco during periods of water deficit. Journal of Experimental Botany 46, 1077–1084.
Kraepiel Y, Rousselin P, Sotta B, Kerhoas L, Einhorn J, Caboche M, Miginiac E. 1994. Analysis of phytochrome-deficient and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia. The Plant Journal 6, 665–672.[CrossRef]
Laby RJ, Kincaid MS, Kim DG, Gibson SI. 2000. The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. The Plant Journal 23, 587–596.[CrossRef][Web of Science][Medline]
LaRosa PC, Hasegawa PM, Rhodes D, Clithero JM, Watad A-EA, Bressan RA. 1987. Abscisic acid stimulated osmotic adjustment and its involvement in adaptation of tobacco cells to NaCl. Plant Physiology 85, 174–181.
Leigh RA, Ahmad N, Wyn Jones RG. 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153, 34–41.[CrossRef][Web of Science]
Leung J, Merlot S, Giraudat J. 1997. The Arabidopsis abscisic acid-insensitive2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. The Plant Cell 9, 759–771.[Abstract]
Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J. 2001. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. The Plant Journal 25, 295–303.[CrossRef][Web of Science][Medline]
Murphy GJP. 1984. Metabolism of R,S-[2-14C]abscisic acid by non-stressed and water-stressed detached leaves of wheat (Triticum aestivum L.). Planta 160, 250–255.[CrossRef]
Ober ES, Sharp RE. 1994. Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. I. Requirement for increased levels of abscisic acid. Plant Physiology 105, 981–987.[Abstract]
Pesci P. 1993. Glucose mimics the enhancing effect of light on ABA-induced proline accumulation in hydrated barley and wheat leaves. Journal of Plant Physiology 142, 355–359.
Pierce M, Raschke K. 1980. Correlation between loss of turgor and accumulation of abscisic acid in detached leaves. Planta 148, 174–182.[CrossRef]
Qin XQ, Zeevaart JAD. 2002. Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance. Plant Physiology 128, 544–551.
Ribaut J-M, Martin HV, Pilet P-E. 1996. Abscisic acid turnover in intact roots: a new approach. Journal of Plant Physiology 148, 761–764.
Rook F, Corke F, Card R, Munz G, Smith C, Bevan MW. 2001. Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling. The Plant Journal 26, 421–433.[CrossRef][Web of Science][Medline]
Savoure A, Hua X-J, Bertauche N, Van Montagu M, Verbruggen N. 1997. Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Molecular and General Genetics 254, 104–109.[CrossRef]
Sharp RE, LeNoble ME. 2002. ABA, ethylene and the control of shoot and root growth under water stress. Journal of Experimental Botany 53, 33–37.
Sharp RE, Wu Y, Voetberg GS, Saab IN, LeNoble ME. 1994. Confirmation that abscisic acid accumulation is required for maize primary root elongation at low water potentials. Journal of Experimental Botany 45, 1743–1751.[Web of Science]
Shinozaki K, Yamaguchi-Shinozaki K, Seki M. 2003. Regulatory network of gene expression in the drought and cold stress responses. Current Opinion in Plant Biology 6, 410–417.[CrossRef][Web of Science][Medline]
Smirnoff N, Cumbes QJ. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060.[CrossRef][Web of Science]
Stewart CR, Morris CJ, Thompson JF. 1966. Changes in amino acid content of excised leaves during incubation. II. Role of sugar in the accumulation of proline in wilted leaves. Plant Physiology 41, 1585–1590.
Strizhov N, Abraham E, Okresz L, Blickling S, Zilberstein A, Schell J, Koncz C, Szababos L. 1997. Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. The Plant Journal 12, 557–569.[CrossRef][Web of Science][Medline]
Suzuki M, Kao C-Y, Cocciolone S, McCarty DR. 2001. Maize VP1 complements Arabidopsis abi3 and confers a novel ABA/auxin interaction in roots. The Plant Journal 28, 409–418.[CrossRef][Web of Science][Medline]
van der Weele CM, Spollen WG, Sharp RE, Baskin TI. 2000. Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media. Journal of Experimental Botany 51, 1555–1562.
Verslues PE, Bray EA. 2004. LWR1 and LWR2 are required for osmoregulation and osmotic adjustment in Arabidopsis thaliana. Plant Physiology 136, 2831–2842.
Verslues PE, Sharp RE. 1999. Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone. Plant Physiology 119, 1349–1360.
Voetberg GS, Sharp RE. 1991. Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiology 96, 1125–1130.
Weyers JDB, Paterson NW, A'Brook R, Peng Z-Y. 1995. Quantitative analysis of the control of physiological phenomena by plant hormones. Physiologia Plantarum 95, 486–495.[CrossRef]
Xiong L, Zhu J-K. 2003. Regulation of abscisic acid biosynthesis. Plant Physiology 133, 29–36.
Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. 1982. Living with water stress: evolution of osmolyte systems. Science 217, 1214–1222.
Zeevaart JAD. 1999. Abscisic acid metabolism and its regulation. In: Hall M, Libbenga K, eds. Biochemistry and molecular biology of plant hormones. Amsterdam: Elsevier Science BV, 189–207.
Zeevaart JAD, Creelman RA. 1988. Metabolism and physiology of abscisic acid. Annual Review of Plant Physiology and Plant Molecular Biology 39, 439–473.[CrossRef][Web of Science]
Zhang J, Davies WJ. 1987. Increased synthesis of ABA in partially dehydrated root tips and ABA transport from roots to leaves. Journal of Experimental Botany 38, 2015–2023.
Zhang J, Nguyen HT, Blum A. 1999. Genetic analysis of osmotic adjustment in crop plants. Journal of Experimental Botany 50, 292–302.
Zhu J-K. 2002. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology 53, 247–273.[CrossRef][Medline]
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