(en)The present invention concerns a transgenic plant with enhanced tolerance to reduced water availability. The plant includes an expression cassette including an abscisic acid responsive element binding factor (ABF), which is capable of expressing the recombinant ABF. A method to improve plant's tolerance to reduced water availability is also provided.

1.ApplicationNumber: US-16901305-A
1.PublishNumber: US-2005244971-A1
2.Date Publish: 20051103
3.Inventor: KIM SOO Y.

4.Inventor Harmonized: KIM SOO Y(KR)

5.Country: US
6.Claims:
(en)The present invention concerns a transgenic plant with enhanced tolerance to reduced water availability. The plant includes an expression cassette including an abscisic acid responsive element binding factor (ABF), which is capable of expressing the recombinant ABF. A method to improve plant's tolerance to reduced water availability is also provided.

7.Description:
(en)This is a Continuation-in-Part application of U.S. Ser. No. 10/128,456 filed on Apr. 24, 2002, which is herein incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
The present invention relates to transgenic plants with enhanced stress tolerance. In particular, the invention relates to the transgenic plants, which express abscisic acid responsive element-binding transcription factors, ABFs, in amounts effective to confer increased tolerance to reduced water availability to the transgenic plants.
Being sessile, plants have the capability to adapt to adverse environmental conditions such as drought, cold and high salt. Under these stress conditions, the plant hormone abscisic acid (ABA) level increases in vegetative tissues, triggering adaptive responses that are essential for their survival and productivity (Leung and Giraudat, 1998; Zeevaart and Creelman, 1988). Under water deficit conditions, for example, ABA induces stomatal closure, minimizing water loss through transpiration. The ABA-controlled process is vital for plant survival, and ABA deficient or ABA response mutants are susceptible to water stress. On the other hand, high levels of ABA inhibit overall plant growth (Himmelbach et al., 1998).
Underlying the ABA-mediated stress responses is the transcriptional regulation of stress responsive gene expression (Giraudat et al., 1994; Busk and Pages, 1998). Numerous genes have been reported that are up-regulated under stress conditions in vegetative tissues (Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1997). These include a class of genes known as LEA (Late Embryogenesis Abundant) genes, which are expressed abundantly in developing seeds under normal conditions, osmolyte biosynthetic genes and genes of general cellular metabolism. In general, the gene products are considered to have protective or adaptive roles under stress conditions. In addition, expression of many regulatory genes including various kinase/phosphatase and transcription factor genes is also induced by abiotic stresses. Not all stress-inducible genes are regulated by ABA. However, a large number of them are also responsive to exogenous ABA and, in many cases, their induction is impaired in ABA deficient mutants. Meanwhile, expression of some genes such as rbcS and CAB genes is suppressed by ABA and stress (Bartholomew et al., 1991; Wang et al., 1996; Weatherwax et al., 1996).
Abscisic acid responsive elements (ABREs) that control the ABA and/or stress responsive gene expression have been determined by numerous studies (Giraudat et al., 1994) and their putative cognate trans-acting factors have been isolated (Busk and Pages, 1998). Most ubiquitous among the cis-elements is a group of sequences sharing the (C/T)ACGTGGC consensus. Many of these elements contain the G-box (CACGTG) sequence (Giuliano et al., 1988), which is also present in numerous genes regulated by other environmental cues (Menkens et al., 1995). Another group of ABREs, known as “coupling element”, “hex3” or “motif III” (Busk and Pages, 1998), shares the CGCGTG core sequence.
Based on their interactions with these two types of ABREs, which will be referred to as “ABREs” hereafter, a number of putative trans-acting factors have been isolated (Busk and Pages, 1998). Also, their homologs and numerous other G-box binding factors, all belonging to the bZIP class proteins (Landschulz et al., 1988), are able to interact with the ABREs in vitro (Foster et al., 1994). However, the evidence showing that any of these biochemically-identified factors play a role in ABA or stress signaling in planta is still lacking.
In an effort to identify transcription factors that control ABA responsive gene expression during vegetative growth, we recently isolated four ABRE binding bZIP factors by yeast one-hybrid screening of an Arabidopsis cDNA expression library (Choi et al., 2000).
Expression of the factors, referred to as ABF1 through ABF4 (ABRE Binding Factors 1-4), is ABA- and stress-inducible, and they can transactivate an ABRE-containing reporter gene in yeast. ABF2 and ABF4 were reported also by Uno et al. (Uno et al., 2000), who named them as AREB1 and AREB2, respectively, and showed that the factors can activate an ABA responsive promoter in protoplasts. In order to investigate ABFs' in vivo functions, we generated transgenic Arabidopsis plants constitutively overexpressing them. Here we show that ABF3 and ABF4 transgenic lines are hypersensitive to ABA and that they exhibit several other ABA/stress-associated phenotypes including enhanced drought tolerance. Amino acid sequence of ABF3 (SEQ ID NO: 1) was disclosed in U.S. Pat. No. 6,218,527 (Apr. 17, 2001), and amino acid sequence of ABF4 (SEQ ID NO: 2) was disclosed in U.S. Pat. No. 6,232,461 (May 15, 2001) by the inventor himself. The expression patterns of the two ABFs correlated well with their overexpression phenotypes.
SUMMARY OF THE INVENTION
The object of the present invention is to provide transgenic plants, which are substantially more tolerant to reduced water availability and other environmental stresses than untransformed plants, the cells of which comprise a recombinant DNA segment encoding an abscisic acid responsive element-binding factor ABF, wherein the recombinant ABF expression is in amounts effective to confer enhanced tolerance to reduced water availability and other stresses to the transformed plants.
Further, the transgenic plants have the recombinant abscisic acid responsive element-binding factor ABF3 and/or ABF4.
The present invention also provides a seed produced by the transgenic plants and a progeny, clone, cell line or cell of the transgenic plant.


BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 . Growth Phenotype of 35S-ABF3 and 35S-ABF4 Plants. (A) Growth on soil. 35S-ABF3 (line A319) and 35S-ABF4 (line A405) transgenic plants were grown for 3 weeks on soil. The inset shows fully-grown siliques, two each from Ler, 35S-ABF3 and 35S-ABF4 plants (from left to right). (B) The relationship between the ABF4 expression level and the severity of growth phenotype. Left, RNA gel blot analysis of ABF4 expression in Ler and 35S-ABF4 transgenic lines, A402, A405 and A406. Lower panel shows the ethidium bromide staining of the RNA gel. Each lane contains 25 μg of total RNA. Right, 35S-ABF4 transgenic lines with varying degrees of ABF4 expression. Only the aerial parts of the plants are shown for clarity. (C) Left, germination of 35S-ABF3 transgenic seeds on ABA-free medium. 10 week-old seeds of Ler, line A333 and line A319 were plated on ABA-free medium after 4 days of cold treatment, and germination (fully emerged radicle) was scored at various times. Each data point represents the means of triplicate experiments (n=50 each). Standard errors are smaller than the symbols. Right, RNA gel blot analysis of ABF3 expression in Ler and transgenic lines A33 and A19. Lower panel shows the ethidium bromide staining of the RNA gel. Each lane contains 25 μg of total RNA. The ABF3 expression levels in A33 and A19 lines are comparable to the ABF4 expression levels in A405 and A406 lines.
FIG. 2 . ABA Sensitivity of 35S-ABF3 and 35S-ABF4 Plants. (A) Growth of transgenic plants on medium containing 0.5 μM ABA. Seeds were germinated and grown for 12 days. (B) Growth of transgenic plants on medium containing 0.25 μM ABA. Seeds were germinated on the medium for 3 days and representative plants are shown. (C) ABA dose-response of germination. Seeds, 6 months old after harvest and pre-chilled at 4° C. for 4 days, were germinated on media containing various concentrations of ABA, and seedlings with fully emerged radicles were counted after 3 days. Experiments were done in triplicates (n=50 each), and the bars show standard errors. (D) ABA dose-response of root growth. Seeds were germinated for 4 days on ABA-free medium and the seedlings (n=6) were transferred to media containing various concentrations of ABA. Root elongation was measured 5 days after the transfer. The experiments were performed more than four times, sometimes using different transgenic lines, and the results were consistent. The small bars represent standard errors. Transgenic lines A319 (ABF3) and A405 (ABF4) were used.
FIG. 3 . Salt and Glucose Sensitivity of 35S-ABF Plants. (A) Salt effect on germination. Seeds of Ler, aba1-1, abi1-1, abi2-1, 35S-ABF3 (line A319) and 35S-ABF4 (line A405) were plated after 4 days of cold treatment on media containing 50, 100, or 150 mM NaCl, and germination (fully open cotyledons) was scored after 4 days. Experiments were done in triplicates (n=50 each), and standard errors are smaller than the symbols. (B) Salt effect on newly germinated seedling growth. Seeds of the same transgenic lines were germinated for 4 days on media containing 100 mM of NaCl, KCl or mannitol, and representative seedlings are shown. (C) Glucose response of 35S-ABF transgenic plants. Seeds were germinated and grown for 14 days on the regular growth medium or the same medium supplemented with 3% glucose or mannitol at vertical position. A319, A333, A405, and A406 indicate transgenic lines.
FIG. 4 . Epinasty and Obstacle-touching Response of 35S-ABF4 Plants. (A) Epinastic curvature of 35S-ABF4 transgenic leaves. Ler and transgenic plants were grown on MS plates for 2 weeks. ABF4 expression level is highest in the A406 line and lowest in the A402 line ( FIG. 1B ). (B) Obstacle-touching response of 35S-ABF4 plants. Plants (line A405) were grown on 1.5% agar plates for 4 days at vertical position and then for 5 days at an angled (45°) position. The arrows indicate the root tip position before changing to the slanted orientation.
FIG. 5 . Drought Tolerance of 35S-ABF3 and 35S-ABF4 Plants. (A) Drought tolerance of 35S-ABF3 transgenic plants (line A319). Transgenic and wild type plants (n=100 each) were grown on soil in the same container for two weeks, withheld from water for 11 days, and then re-watered. The picture was taken 3 days after the rewatering. (B) Drought tolerance of 35S-ABF4 transgenic plants (line A405). Plants at similar developmental stages (two weeks old wild type plants and 3 weeks old ABF4 plants) were withheld from water for 12 days and then re-watered. The picture was taken 3 days after the re-watering. (C) and (D) show the transpiration rates of 35S-ABF3 and 35S-ABF4 transgenic plants, respectively. Leaves of similar developmental stages were excised and weighed at various times after the detachment. Each data point represents the mean of duplicate measurements (n=9 each). Standard errors are smaller than the symbols. (E) Stomatal aperture of ABF transgenic plants (lines A319 and A405). Stomatal guard cells were observed in the middle of watering period. Arrows indicate guard cells, and the insets show representative stomata.
FIG. 6 . Expression of ABA-regulated Genes in 35S-ABF Transgenic Lines. RNA levels of ABA responsive genes were determined by RT-PCR, using total RNAs isolated from 2 week old plants grown on MS plates. Lines A319 and A405 represent transgenic lines with higher ABF expression, whereas lines A325 and A402 represents transgenic lines with lower ABF expression.
FIG. 7 . Expression Patterns of ABF3 and ABF4. (A) Tissue-specificity of ABF3 and ABF4 expression. RNA was isolated from various tissues of wild type plants grown under normal condition and the expression of ABF3 and ABF4 was determined by RT-PCR. L, leaves from three-week-old plants. R, roots from three-week-old plants. F, flowers. Si, immature siliques. (B) and (C) Histochemical GUS staining of the ABF3 and the ABF4 promoter activity, respectively. T3 homozygous plants were stained with X-gluc for 6 (panels e and f) or 24 hrs (other panels). a, 2-day-old seedlings. Insets show embryos from immature siliques (top) or dry seeds (bottom). b. 5 day-old seedlings. The arrow in (B) shows the newly emerging shoot. c. 2-week-old seedlings. d. 2-week-old seedlings treated with 100 μM ABA (ABF3) or 200 mM NaCl (ABF4). e, root tips. The left half of the panel in (C) shows a lateral root primordium. f, guard cells. g, flowers. h, siliques. Arrows indicate the silique abscission zone.


DETAILED DESCRIPTION OF THE INVENTION
Growth Phenotypes of ABF3 and ABF4 Overexpression Lines
In order to investigate the in vivo functions of ABF3 and ABF4, we employed an overexpression approach. The coding region of ABF3 or ABF4 was fused to the cauliflower mosaic virus 35S promoter and each construct was used to transform Arabidopsis (ecotype Ler) plants. Thirtyeight and twelve T3 homozygous lines were recovered from the 35S-ABF3 and the 35S-ABF4 constructs, respectively, and, after preliminary analysis, transgenic lines with higher ABF expression levels were selected for more detailed analysis.
Compared with wild type plants, 35S-ABF3 transgenic plants exhibited mild growth retardation in the aerial parts; petioles were slightly shorter and leaves were rounder in shape ( FIG. 1A ). The degree of retardation was not severe, however, and overall growth patterns were similar to wild type plants except that siliques were somewhat shorter and thicker ( FIG. 1A , inset). In contrast, 35S-ABF4 transgenic plants exhibited severe growth retardation ( FIG. 1A ), which was dependent on the ABF4 expression level ( FIG. 1B ). Petioles were shorter, leaves were smaller, flowering was delayed, and plants were shorter. Also, germination of 35S-ABF3 plants was delayed several hours compared with that of wild type plants in the absence of ABA ( FIG. 1C ). 35S-ABF4 plants, on the other hand, germinated normally (data not shown), and, thus, the growth retardation observed with 35S-ABF4 plants was a post-germination process.
ABA Response of 35S-ABF Plants
To test whether ABF3 or ABF4 overexpression affected ABA sensitivity, 35S-ABF3 and 35S-ABF4 transgenic plants were germinated and grown on media containing various concentrations of ABA. When ABA concentration was 0.5 μM or above, the growth of 35S-ABF plants was completely arrested after radicles emerged, i.e., cotyledon greening/expansion and root growth were severely inhibited and none of the transgenic seedlings developed to have true leaves ( FIG. 2A ). At the same conditions, wild type plants continued to grow and develop although at slower rates than on ABA-free medium. The ABA hypersensitivity of 35S-ABF transgenic plants was also observed at 0.25 μM ABA ( FIG. 2B ), although the transgenic seedlings eventually grew to have true leaves (data not shown).
To see the stage-specificity of ABA response, ABA dose-response was examined during and after germination. As shown in FIG. 2C , 35S-ABF transgenic plants were hypersensitive to ABA at the germination stage, 0.5 μM ABA being sufficient to inhibit their germination efficiencies to 8 (ABF3) or 28% (ABF4) while wild type plants retained 80% germination at the same condition. Likewise, root growth of the 35S-ABF transgenic lines was hypersensitive to ABA ( FIG. 2D ). At 0.5 μM ABA, wild type root growth was 84% of its control rate, whereas those of 35S-ABF4 and 35S-ABF3 plants were 35% and 51% of their control rates, respectively. At 1 μM ABA, root growth of the 35S-ABF4 plants reduced to 6% and the 35S-ABF3 plants to 37%. In addition, lateral root and aerial part growth of 35S-ABF transgenic plants was significantly inhibited at this concentration (data not shown). Wild type plants grew at 62% of the control rate at the same ABA concentration. Transgenic root growth was almost completely arrested when ABA concentration was above 5 μM, whereas wild type plants still continued to grow. The results indicate that both germination and post-germination growth of 35S-ABF transgenic plants are hypersensitive to ABA.
Salt Response of 35S-ABF Plants
High concentrations of salts inhibit the germination of Arabidopsis (Werner and Finkelstein, 1995; Leon-Kloosterziel et al., 1996; Quesada et al., 2000; Zhu, 2000). Several studies show that ABA plays a role in the inhibition process. Although not all salt-insensitive mutants are ABA-insensitive (Warner and Finkelstein, 1995), all ABA deficient (aba) and ABA insensitive (abi) mutants exhibit salt-insensitivity during germination (Leon-Kloosterziel et al., 1996). This is probably because ABA, whose level increases by high salt, promotes the inhibition process. Since the expression of both ABF3 and ABF4 is salt-inducible (Choi et al., 2000), they may participate in salt response, i.e., salt-induced germination inhibition in this case. To test this, 35S-ABF transgenic plants were germinated on media containing various concentrations of NaCl. FIG. 3A shows that the germination of wild type and aba1, abi1 and abi2 mutant plants was not affected by NaCl below 100 mM. In contrast, germination and growth of the 35S-ABF plants were significantly affected by 100 mM NaCl ( FIG. 3 , A and B); radicle emergence, root growth and cotyledon opening/expansion were severely inhibited. In a parallel experiment, the transgenic plants responded to KCl in a similar way to NaCl ( FIG. 3B ). On the other hand, their response to the same concentration or twice the concentration (data not shown) of mannitol, which gives the same osmotic pressure, was normal. Thus, in contrast to the salt-insensitive phenotype of ABA deficient or ABA insensitive mutants, ABF3 and ABF4 overexpression both resulted in salt hypersensitivity at the germination/young seedling stage, and the hypersensitivity appeared to be ionic rather than osmotic in nature.
Sugar Response of 35S-ABF Plants
At higher concentrations, sugars inhibit the development of young seedlings, i.e., they inhibit cotyledon greening/expansion and shoot growth (Jang et al., 1997). According to works done by others, ABA plays an essential role in the glucose or sucrose signal transduction (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000). For example, the ABA-deficient, aba2 mutation is allelic to the sugar insensitive, sis4 mutation, and the glucose or sugar insensitive mutations, gin6, sis5 and sun6 are allelic to the ABA insensitive mutation abi4. Also, the studies show that other aba mutants and, to some degree, abi5 mutants are insensitive to glucose. Thus, ABF overexpression might have affected sugar sensitivity as well, if indeed ABF3 and ABF4 mediate ABA signaling. We addressed this by examining their response to glucose, which exerts more severe growth inhibition than other sugars (Jang et al., 1997). Under our experimental conditions, wild type seedlings showed growth defects such as the inhibition of cotyledon greening and true leaf development when glucose concentration was above 4% (data not shown). The aerial part growth of 35S-ABF transgenic lines, on the other hand, was arrested completely at 3% glucose, at which wild type plants developed fully ( FIG. 3C ). Thus, 35S-ABF transgenic plants were hypersensitive to glucose. This enhanced response of the transgenic plants was not observed with the same concentration of mannitol, which inhibited the growth of both wild type and 35S-ABF transgenic plants significantly but similarly. Thus, the hypersensitivity was glucose-specific rather than osmotic.
Epinasty and Obstacle-Touching Response of 35S-ABF4 Plants
Recent genetic studies show that ABA signaling pathways interact with those of ethylene. According to the studies, ABA-mediated inhibition of germination is negatively regulated by ethylene, whereas ethylene signaling components are required for the ABA inhibition of root growth (Beaudoin et al., 2000; Ghassemian et al., 2000). The involvement of auxin in ABA-dependent stress response has also been demonstrated (Vartanian et al., 1994). When exposed to progressive drought, new lateral roots take a short and tuberized form. The process, known as ‘drought rhizogenesis’, is impaired not only in the ABA deficient aba-1 and ABA-insensitive abi1-1 mutants but also in the auxin resistant axr1-3 mutant. To test whether the overexpression of ABF3 or ABF4 affected ethylene or auxin sensitivity, we compared the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and indole-3-acetic acid (IAA) on the growth of 35S-ABF and wild type plants. We did not see any differences in their responses. However, leaves of 35S-ABF4 plants have a tendency to have epinastic curvature when grown on plates ( FIG. 4A ), which is generally attributed to ethylene action under stress conditions (Jackson, 1997). Also, roots of 35S-ABF4 transgenic plants exhibited abnormality in the obstacle-touching response (Okada and Shimura, 1990; Simmons et al., 1995). As shown in FIG. 4B , roots of wild type plants grow in a wavy pattern when grown on a hard agar surface at an inclined position. The wavy pattern of root growth, however, was significantly diminished in 35S-ABF4 plants. The obstacle-touching response is impaired in several auxin resistant mutants, indicating that auxin signalling components are involved in the process (Okada and Shimura, 1992). Thus, the result implies that auxin signaling pathway(s) might have been perturbed in 35S-ABF4 plants. Taken together, our results suggest that ABF4 overexpression might have affected some aspects of ethylene and auxin responses.
Drought Tolerance of 35S-ABF Plants
One of the key ABA-controlled processes is the stomatal closure under water stress conditions, which minimizes water loss through transpiration (Leung and Giraudat, 1998). ABA biosynthesis mutants and some of the ABA response mutants (i.e., abi1 and abi2), therefore, are very susceptible to drought because of the impaired stomatal aperture regulation (Leung and Giraudat, 1998; Schroeder et al., 2001). Thus, ABF3 and ABF4 overexpression lines are expected to exhibit altered response to water deficit conditions if the factors are involved in ABA/stress signaling. To address this, we examined the drought tolerance of 35S-ABF plants. As shown in FIG. 5A , wild type plants withered completely when withdrawn from water for 11 days and only 16% of them survived to maturity when re-watered afterwards. 35S-ABF3 plants, however, were not affected noticeably and all survived the treatment to set seeds. In a similar experiment, 35S-ABF4 plants also exhibited higher survival rates under water deficit condition; all of them survived a 12 day drought treatment, whereas 33% of the wild type plants survived to set seeds ( FIG. 5B ). Thus, both 35S-ABF3 and 35S-ABF4 plants survived the drought conditions better than wild type plants.
The enhanced drought tolerance of the transgenic plants could be attributed, at least in part, to their lower transpiration rates. When measured by the fresh weight loss of detached rosette leaves, the water loss rates of 35S-ABF3 and 35S-ABF4 transgenic lines were less than half and approximately 70% of the wild type plants', respectively ( FIG. 5 , C and D). Consistent with the result, the stomata of the transgenic plants had smaller openings than wild type plants ( FIG. 5E ). Under normal growth condition, ca. 85% (89/104) of the wild type stomata were open, whereas approximately 20% of them were open in 35S-ABF3 (15/71) and 35S-ABF4 (17/71) plants when observed in the middle of watering period. Thus, constitutive overexpression of ABF3 or ABF4 resulted in partial stomatal closure, reduced transpiration and enhanced drought tolerance.
Expression of ABA Responsive Genes in 35S-ABF Plants
To investigate the transcriptional regulatory roles of ABF3 and ABF4 in planta, expression of various ABA/stress responsive genes in 35S-ABF plants was determined. As shown in FIG. 6 , the transcript levels of a number of ABA-regulated genes (Group I) were enhanced in 35S-ABF3 and 35S-ABF4 transgenic lines. These include LEA class genes rd29B (Yamaguchi-Shinozaki and Shinozaki, 1994) and rab18 (Lang and Palva, 1992), whose expression is induced by ABA and abiotic stresses. Expression of the ABA-inducible, cell cycle regulator gene ICK1 (cyclin-dependent kinase inhibitor) (Wang et al., 1998) was also elevated in the 35S-ABF transgenic lines. The ICK1 gene has been suggested to mediate cell division arrest by ABA. In the 35S-ABF3 lines, strong enhancement of ABI1 (Leung et al, 1994; Meyer et al., 1994) RNA level was observed, and ABI2 (Leung et al., 1997) transcript level was elevated albeit the degree of increase was lower. Increase in the ABI1 RNA level was also observed in the 35S-ABF4 line with higher ABF4 expression (A405). Expression of ABI1 and ABI2, which encode homologous protein phosphatase 2Cs and whose mutations result in defective stomatal closing and wilty phenotype (Schroeder et al., 2001), is enhanced by ABA and water stress (Leung et al., 1997). Meanwhile, the RNA level of the ABA-repressible gene SKOR (Gaymard et al., 1998) was significantly reduced or undetectable in 35S-ABF4 transgenic lines (Group II). The gene encodes a root-specific K + outward rectifying channel, and ABA repression of its expression has been suggested to be a part of adaptive water stress response. Similarly, guard cell ion channel genes KAT1 and KAT2 (Anderson et al., 1992; Pilot et al., 2001) were negatively regulated in the 35S-ABF3 line with higher ABF3 level (A319). The two ion channels normally mediate K + influx, enabling stomatal opening, but their activity is inhibited by ABA. Also, stress responsive biosynthetic genes (Group III), chalcone synthase gene CHS (Feinbaum and Ausubel, 1988) and alcohol dehydrogenase gene ADH1 (de Bruxelles et al., 1996), were down-regulated. The transcript levels of the two genes were, however, higher in the 35S-ABF transgenic lines when plants were treated with high salt, suggesting that stress-induced post-translational modification of ABF3 and ABF4 may be required for the regulation of these genes. In summary, overexpression of ABF3 or ABF4 resulted in the modulation of ABA/stress responsive gene expression and the two factors played positive or negative roles depending on specific genes and environmental conditions.
Expression Patterns of ABF3 and ABF4
The phenotypes described so far indicated that overexpression of ABF3 or ABF4 enhanced various aspects of ABA response. To assess the physiological relevance of the results and to obtain further clues about their functions, we investigated their temporal and spatial expression patterns. We first investigated the tissue-specificity of their uninduced, basal expression. Since the basal expression levels of ABF3 and ABF4 are very low (Choi et al., 2000), we employed the coupled reverse transcription and polymerase chain reaction (RT-PCR) for the analysis. As shown in FIG. 7A , relatively higher expression of both ABF3 and ABF4 was detected in roots. Also, lower ABF3 expression was observed in flowers but not in leaves and siliques at the same condition. On the other hand, weak ABF4 expression was detectable in leaves, flowers and siliques.
More detailed temporal and spatial expression patterns of ABF3 and ABF4 were determined by histochemical GUS staining of transgenic plants that harboured an ABF promoter-GUS reporter construct. With the ABF3 promoter (2.1 kb) construct, GUS activity was undetectable in embryos, but it was observed in the emerging radicles at the germination stage ( FIG. 7B , a) and in most of the vegetative tissues at later stages ( FIG. 7B , b and c). Roots were stained most strongly except the tip area, and petioles, leaf vascular tissues and guard cells exhibited relatively strong GUS activity ( FIG. 7B , b-f). Emerging shoots and younger leaves ( FIG. 7B , b and c), on the other hand, exhibited GUS staining only after ABA, salt or mannitol treatment ( FIG. 7B , d). In mature plants, GUS staining was detected also in anthers, stigma, and siliques (abscission zone, replum, and funiculi) ( FIG. 7B , g and h).
The ABF4 promoter was active in embryos from green siliques ( FIG. 7C , a), but its activity decreased as embryos became mature and was not detected in newly germinated seedlings except in some limited regions (radicle tip, shoot meristem region, and cotyledon tips) ( FIG. 7C , a). At later stages, starting from the stage of fully-expanded cotyledons ( FIG. 7C , b), ABF4 promoter activity was observed in all vegetative tissues ( FIG. 7C , c-f), and also in floral organs and siliques (abscission zone, replum, and funiculi) ( FIG. 7C , g and h). The ABF4 promoter was most active in roots, especially in the growing regions (meristem, elongation zone and lateral root primordia) ( FIG. 7C , e), suggesting its role in growth regulation. Also, it exhibited strong activity in petioles and guard cells ( FIG. 7C , c and f). Salt treatment of seedlings enhanced the ABF4 promoter activity somewhat ( FIG. 7C , d).
DISCUSSION
ABA-regulated gene expression plays a central role in ABA signaling, and numerous ABA/stress responsive genes are regulated by the (C/T)ACGTGGC- or CGCGTG-containing. ABREs. Thus, identifying relevant transcription factors is critical for the delineation of ABA signal transduction cascades. Many studies show that ABA signaling pathways are tissue-specific (Leung and Giraudat, 1998; Giraudat et al., 1994), and several seed-specific ABA signaling components (ABI3, ABI4, and ABI5) have been identified by genetic screens. ABI3 and ABI4 encode transcription factors (Giraudat et al., 1992; Finkelstein et al., 1998), whose binding sites and immediate target genes are unknown. Recently, ABI5 has been shown to encode a bZIP factor that belongs to a seed-specific subfamily of ABF-related factors (Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000) and its role in postgermination developmental arrest has also been demonstrated (Lopez-Molina et al., 2001). However, ABRE binding factors whose major function is to mediate the ABA signaling during vegetative growth have not been reported, although numerous bZIP factors are known to interact with the ABREs in vitro (Foster et al., 1994).
ABFs are unique among the ABRE binding bZIP factors in that, unlike most of the other plant bZIP factors, they can interact with both the G-box type and the CGCGTG-containing ABREs (Choi et al., 2000). The broad binding specificity, together with their transactivation capability of an ABRE-containing reporter gene and the stress-inducibility of their expression, suggested that ABFs have a potential to regulate a large number of ABA/stress responsive genes and, thus, are likely to participate in stress responsive ABA signaling. In order to address this question, we employed an overexpression approach. Considering the potential functional redundancy of ABFs and numerous other bZIP factors interacting with ABREs (Foster et al., 1994), the approach would be a better way than loss of function approaches such as antisense, knockout and RNA interference. The overexpression of ABFs (we estimate that ABF3 and ABF4 levels in the 35S-ABF transgenic lines used in our study range from approximately 2 to 10 folds of their ABA-induced levels), however, might have caused non-natural conditions. For example, genes that are not normally regulated by ABFs might have been turned on or off. Also, it may have affected the functions of other members of ABFs or potentially other bZIP factors, by titrating them out via non-natural heterdimerization. Thus, the overexpression phenotypes need to be interpreted with caution, and their roles can be further confirmed by other experimental means. Nevertheless, our results show that ABF3 or ABF4 overexpression conferred several ABA-associated phenotypes such as ABA hypersensitivity, sugar hypersensitivity and enhanced drought tolerance, with altered expression of ABA/stress responsive genes. Thus, our data provide a strong in vivo case for the involvement of ABF3 and ABF4 in stress responsive ABA signaling.
Whereas the ABA hypersensitivity conferred by ABF3 or ABF4 overexpression was very distinct and observed both at the germination and at later growth stages ( FIG. 2 ), the overexpression effects on growth in the absence of exogenous ABA were either moderate (ABF3) or developmental stage-dependent (ABF4) ( FIG. 1 ). ABF3 exerted inhibitory effect on both germination and seedling growth. However, the low degree of inhibition compared with that in the presence of exogenous ABA suggests that ABF3 alone is not sufficient for the inhibitory function. On the other hand, ABF4 overexpression had little effect on germination but had a severe effect on seedling growth, suggesting that ABF4 activity is developmentally modulated. Alternatively, the result may indicate that ABA inhibition of seedling growth is mediated by a mechanism that differs from the germination inhibition mechanism. Also, the developmental stage-dependency of the ABF4 effects implies that the growth retardation of 35S-ABF4 plants result probably from the constitutive operation of a part of ABA signal transduction cascades rather than from the pleiotropic effects of ABF4 overexpression. Except the varying degrees of growth retardation, neither 35S-ABF4 nor 35S-ABF3 plants did show abnormality in general development. Thus, their overexpression affected growth rate, but not developmental processes.
Other ABA- or stress-associated phenotypes of 35S-ABF3 and 35S-ABF4 transgenic plants include their hypersensitivities to salt and glucose, and 35S-ABF4 plants exhibited additional phenotypes (i.e., epinasty of leaves and abnormal obstacle-touching response) that can be related to altered ethylene or auxin response. The salt and glucose hypersensitivities may reflect the increased sensitivity to high osmolarity, since both high salt and high sugar accompany increase in osmolarity. However, 35S-ABF plants responded normally to mannitol, indicating that osmotic sensitivity was not affected. Thus, it appears that ABF3 and ABF4 are involved only in the non-osmotic branches of salt and glucose signaling pathways. The sugar-mediated developmental arrest is confined to a narrow window of developmental stages, approximately 2 days postgermination, and is mediated by increased ABA level via an ABI4-dependent signaling cascade (Gazzarrini and McCourt, 2001). Also, it has been reported that ABA mediates developmental arrest at the similar stage and that the inhibition process requires ABI5 (Lopez-Molina et al., 2001), whose mutations result in weak sugar insensitivity. Thus, our results suggest that ABF3 and ABF4 have overlapping functions with ABI4 and ABI5 in mediating the sugar- and ABA-induced developmental arrest. This is particularly so in the case of ABF3, since the onset of its expression coincides with the early developmental stage ( FIG. 7B , a).
Stomatal closure is a key ABA-controlled process in coping with water deficit conditions. Our data indicate that ABF3 and ABF4 are involved in this process. Their overexpression resulted in lower transpiration and enhanced drought tolerance ( FIG. 5 ), which are reminiscent of the phenotypes of the ABA hypersensitive mutant era1 (Pei et al., 1998). Furthermore, the stomatal openings of 35S-ABF transgenic plants were smaller than those of wild type plants, and altered expression of several genes involved in stomatal aperture regulation has been observed in the transgenic plants ( FIG. 6 ). Among the genes we investigated, ABI1 was the most strongly affected, especially in ABF3 overexpression lines. ABI1 is known to be a negative regulator of ABA signaling (Gosti et al., 1999), although its expression is enhanced by ABA and high osmolarity while reduced in the aba1 and abi1 mutants (Leung et al., 1997). It is not clear, thus, whether the increased ABI1 level played a positive or a negative role in the stomatal closing. Whatever the ABI1's role might be, our results indicate that ABI1 expression is subject to ABF3 regulation and that ABF3 and ABF4 overexpression affected the expression of genes involved in stomatal movement and/or guard cell ABA signaling (Schroeder et al., 2001), the net result being enhanced stomatal closure.
The transcript level changes of ABA responsive genes in 35S-ABF transgenic lines demonstrate that ABF3 and ABF4 function as transcriptional regulators in planta. As shown in FIG. 6 , both positive and negative changes were observed depending on specific genes. The result suggests that different subsets of ABF3 and ABF4 target genes are regulated via different mechanisms. Also, the negative regulation of some genes under normal growth condition but positive regulation of the same genes after salt treatment ( FIG. 6 , Group III) suggest that stress-induced modification of ABFs activities is required for the regulation of these genes. The modifying activity may be limiting under normal condition. Thus, the negative regulation of some genes can be explained, for example, by the binding of transcriptionally inactive, unmodified ABFs, whose proportion increases with higher ABF levels. The modification may involve phosphorylation of ABFs. Involvement of kinase/phosphatases in ABA/stress signalling has been well known (Leung and Giraudat, 1998), and several phosphorylation sites are highly conserved among ABFs (Choi et al., 2000). More recently, Uno et al. (Uno et al., 2000) reported an ABA-activated kinase activity in cultured cells that phosphorylates AREB1 (ABF2) and AREB2 (ABF4). Alternatively, it may involve interaction with other regulatory proteins.
The expression patterns of ABF3 and ABF4 were consistent with their functions suggested by their overexpression phenotypes. Spatially, both promoters were most active in roots and guard cells, consistent with their roles during water stress response. Also, ABF3, which exerted only minor growth inhibition, was weakly expressed in the growing tissues (root tips, new shoots, and new leaves), whereas ABF4 was strongly expressed in the growing regions of roots (meristem, elongation zone and lateral root primordia). Temporally, strong ABF3 promoter activity was observed in the newly germinated seedlings, consistent with its more pronounced effect on the germination ( FIGS. 1C and 2C ). On the other hand, the ABF4 promoter exhibited major activity at the onset of seedling growth, in agreement with its severe effect on seedling growth. Our results also show that both ABF3 and ABF4 might function during reproductive stages and seed abscission. Both promoters exhibited strong activity in the abscission zone, replum and funiculi of siliques, and relatively strong activity was also detected in stigma and anthers.
The isolation of multiple factors with similar binding activities but with different expression patterns suggested that the ABA/stress signaling involving the ABREs is likely to be mediated by multiple factors (Choi et al., 2000). Our current results further support the observation. Although their overexpression phenotypes and expression patterns were similar, ABF3 and ABF4 were different from each other in several respects. The details of the temporal and spatial expression patterns differed ( FIG. 7 ). Growth retardation, root growth inhibition and impaired stimulus-touching response were more prominent in ABF4 transgenic lines. Also, minor differences were observed at the molecular level. ABI1 and ABI2 expression levels were higher in 35S-ABF3 transgenic lines, while down-regulation of SKOR expression was observed only in 35S-ABF4 plants. The function of other ABFs, ABF1 and ABF2, remains to be determined, but their roles appear to be quite different from each other and also from ABF3 and ABF4 according to our preliminary data. Thus, ABRE-dependent ABA/stress signaling in vegetative tissues appears to be mediated by multiple factors with overlapping, but distinct functions.
Our data presented and discussed above indicate that ABF3 and ABF4 are involved in stress-responsive ABA signaling. In particular, overexpression of ABF3 and ABF4 clearly improved the survival rate of Arabidopsis plants under drought conditions. In our experiments, we used a constitutive promoter (i.e, 35S promoter). However, other promoters, which are tissue-specific or inducible, also can be employed to drive the expression of ABF genes in transgenic plants. Inducible promoters that are active only under stress conditions will be particularly effective in the case of ABF4, whose constitutive overexpression results in growth retardation. Also, it will be possible to alter stress tolerance of plants by decreasing the expression levels of ABFs or its homologs employing established technologies such as antisense, RNA interference and knockdown.
ABA controls not only the drought response but also responses to other environmental stresses such as high salt (Shinozaki and Yamaguchi-Shinozaki, 1997), cold/freezing (Llorente et al, 2000), heat (Larkindale J, Knight M R., 2002) and wounding (Giraudat et al, 1994). Thus, ABFs can be used to develop transgenic plants that are more tolerant than untransformed plants to these multiple stresses. Furthermore, the cis-regulatory elements which control stress-responsive gene expression and to which ABFs bind are highly conserved among various plants, in both monocot and dicot plants. Thus, overexpression of ABFs in plants other than Arabidopsis will also enhance their drought and other stress tolerance, and it will be possible to develop transgenic vegetable or cereal crop plants with enhanced stress tolerance by expressing ABF genes in them.
EXAMPLES
Arabidopsis Growth
Arabidopsis thaliana ecotype Landsberg erecta (Ler) was used in this study. aba1-1, abi1-1, and abi2-1 seeds (Koornneef et al., 1982, 1984) were obtained from the Arabidopsis Biological Resource Center at the Ohio State University, and their phenotypes were confirmed before use.
Plants were grown at 22° C. under long day condition (16 hr light/8 hr dark cycle) aseptically or on soil. For soil growth, seeds were sown on 1:1:1 mixture of vermiculite, perlite and peat moss irrigated with 0.1% Hyponex, placed at 4° C. for 4 days in the dark to break residual dormancy, and transferred to normal growth conditions. Unless otherwise stated, the plants were watered once a week. For aseptic growth, seeds were treated with 70% ethanol for 5 min and then with 30% household bleach for 5 min, washed 5 times with sterile water, and plated on MS medium (Murashige and Skoog, 1962) solidified with 0.8% Phytoagar. The MS medium was supplemented with 1% sucrose and, as described in the text, was also supplemented with ABA, salts, glucose, or mannitol as needed. For germination test, seeds collected at the same or similar times were used. For root growth measurement, plants were germinated and grown at vertical position.
Constructs and Arabidopsis Transformation
The 35S promoter-ABF coding region constructs (35S-ABF3 and 35S-ABF4) were prepared by replacing the β-glucuronidase (GUS) coding region of pBI121 (Jefferson et al., 1987) with the coding region of ABF3 or ABF4. The GUS sequence was removed after BamHI-SacI digestion, and, after T4 DNA polymerase treatment to remove the 3′overhang, the remaining portion of pBI121 was ligated with the ABF3 or the ABF4 coding region, which was prepared by polymerase chain reaction (PCR) followed by BamHI digestion. The ABF coding regions included their entire coding regions with the stop codons and a BamHI linker sequence was attached in front of the initiation codons for the cloning purpose. The ABF promoter-GUS reporter fusions were prepared by inserting 2.1 kb (ABF3) or 1.2 kb (ABF4) of their 5′ flanking sequences from the initiation codons in front of the GUS reporter gene of pBI101.2 (Jefferson et al., 1987). The promoter fragments were prepared by PCR using Arabidopsis (ecotype, Columbia) genomic DNA as a template and primer sets, 5′-caaacttaccctgttgttgcaact-3′ (SEQ ID NO: 3) and 5′-ctagtctagaaggatcaagcttctggatatttac-3′ (SEQ ID NO: 4) for ABF3, and 5′-gatcaatttgaatttttgatatacatc-3′ (SEQ ID NO: 5) and 5′-ctagtctagattcaatgaaaacaaagcatccaag-3′ (SEQ ID NO: 6) for ABF4. The PCR fragments were digested with XbaI and ligated with the pBI101.2, which was prepared by HindIII digestion followed by Klenow treatment and XbaI digestion. DNA manipulation was according to the standard procedures (Ausubel et al., 1994; Sambrook et al., 1989), and the intactness of the ABF coding regions and the junction sequences was confirmed by DNA sequencing.
Transformation of Arabidopsis was according to the vacuum infiltration method (Bechtold and Pelletier, 1998), using A. tumefaciens strain GV3101. For the phenotypic investigation, T3 or T4 homozygous lines were used. GUS staining patterns were confirmed by observing at least 5 different transgenic lines and T3 homozygous lines were used for detailed analysis.
RNA Isolation, RT-PCR and RNA Gel Blot Analysis
RNA was isolated by the method of Chomczynski and Mackey (1995), with a minor modification (Choi et al., 2000). RNA gel blot analysis and RT-PCR were performed as described (Choi et al., 2000) with following modifications. For RNA gel blot analysis, hybridization was performed at 65° C. in the Rapid-hyb buffer from Amersham Pharmacia Biotech. Exposure time was 6 (ABF3) or 20 hr (ABF4). RT-PCR was performed using the Access RT-PCR System from Promega or the SUPERSCRIPT™ One-Step RT-PCR System from GIBCO BRL. Each RT-PCR result was confirmed by several independent reactions, and free of DNA contamination in RNA preparations was confirmed by using primer sets spanning introns whenever possible. Primers used in the RT-PCR reactions are presented in Table 1.
TABLE 1 RT-PCR Primers Gene Name Sequence (5′ to 3′) actin F: cat cag gaa gga ctt gta (SEQ ID NO: 7) cgg R: gat gga cct gac tcg tca (SEQ ID NO: 8) tac ABF3 F: aga acc tca acc ggt gga (SEQ ID NO: 9) gag tg R: gga gtc aga tca ggt gac (SEQ ID NO: 10) atc tgg ABF4 F: aac tgt gtt caa cag atg (SEQ ID NO: 11) ggt cag R: ggt tcc tcc gta act agc (SEQ ID NO: 12) taa tcc rd29B F: gtg aag atg act atc tcg (SEQ ID NO: 13) gtg gtc R: gcc taa ctc tcc ggt gta (SEQ ID NO: 14) acc tag rab18 F: atg acg agt acg gaa atc (SEQ ID NO: 15) cga tgg R: tat gta tac acg att gtt (SEQ ID NO: 16) cga agc ICK1 F: acg cac acg taa cct aaa (SEQ ID NO: 17) tcg R: gca tct ccg tca tca att (SEQ ID NO: 18) tcg ABI1 F: tca aga ttc cga gaa cgg (SEQ ID NO: 19) aga tc R: gag gat caa acc gac cat (SEQ ID NO: 20) cta ac ABI2 F: gtt ctt gtt ctg gcg acg (SEQ ID NO: 21) gag c R: cca tta gtg act cga cca (SEQ ID NO: 22) tca ag rd29A F: gat aac gtt gga gga aga (SEQ ID NO: 23) gtc ggc R: cag ctc agc tcc tga ttc (SEQ ID NO: 24) act 4acc SKOR F: atg gga ggt agt agc ggc (SEQ ID NO: 25) ggc R: gat tct ctg gta atc ccc (SEQ ID NO: 26) tga ag KAT1 F: ttc tgc gtc gag gaa tac (SEQ ID NO: 27) aat ata g R: ctt agg gtc aac tag aag (SEQ ID NO: 28) ata g KAT2 F: aca caa gac caa tgt caa (SEQ ID NO: 29) tct ctt g R: gtc gac tag aag ata tga (SEQ ID NO: 30) gtg gc ADH1 F: tcc acg tat ctt cgg cca (SEQ ID NO: 31) tg R: tag cac ctt ctg cag cgc (SEQ ID NO: 32) c CHS F: tca cca aca gtg aac aca (SEQ ID NO: 33) tga cc R: gag tca agg tgg gtg tca (SEQ ID NO: 34) gag g F: forward primer, R: reverse primer

Histochemical GUS Staining

In situ assay of GUS activity was performed as described by Jefferson et al. (1987). Whole plants were immersed in 1 mM X-gluc (5-bromo-4-chloro-3-indolyl-β-glucuronic acid) solution in 100 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 0.5 mM ferricyanide, 0.5 mM ferrocyanide, and 0.1% Triton X-100, and after applying vacuum for 5 min, incubated at 37° C. for indicated times. Chlorophyll was cleared from the plant tissues by immersing them in 70% ethanol.
Drought Treatment and Measurement of Transpiration Rate
For drought treatment, 3 week-old, soil-grown plants were withheld completely from water for the specified times. To minimize experimental variations, same numbers of plants were grown on the same tray. With 35S-ABF4 plants, which show growth retardation, two batches of plants, one of the same age and the other of similar developmental stages (i.e., wild type seeds were sown 7 days later so that they were at the similar developmental stages at the end of the treatment) were tested, and similar results were obtained. The whole test was repeated at least four times, sometimes using different arrangements of plants (i.e., test plants on different containers etc.), and the results were consistent. Transpiration rate of detached leaves was measured by weighing freshly harvested leaves placed abaxial side up on open petri dishes on the laboratory bench. Leaves of similar developmental stages (third to fifth true rosette leaves) from three week-old, soil-grown plants were used.
Guard Cells
To examine guard cells, leaves were excised from 3 week-old, soil-grown plants in the middle of watering (3 days after watering) and light periods. Leaves of similar developmental stages (third to sixth true rosette leaves) from 20 different plants of wild type and transgenic lines, respectively, were placed on slides abaxial side up immediately after excision and pictures were taken. The number of guard cells was then counted in the randomly chosen fields, usually 6-7.
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