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Plant Cell. Nov 2002; 14(11): 2723–2743.
PMCID: PMC152723

A Unique Short-Chain Dehydrogenase/Reductase in Arabidopsis Glucose Signaling and Abscisic Acid Biosynthesis and Functions

Abstract

Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA.

INTRODUCTION

Plant growth and development is governed by signaling networks that connect inputs from environmental cues, hormone signals, and nutrient status. Recent studies have suggested a pivotal role of sugars as signaling molecules in plants that integrate external environmental conditions and other nutrients with intrinsic developmental programs modulated by multiple plant hormones (Smeekens, 2000; Coruzzi and Zhou, 2001; Finkelstein et al., 2002; Rolland et al., 2002). How sugar signals influence vital processes from germination, seedling development, root and leaf differentiation, and senescence to stress tolerance remains mostly unknown. Cellular and transgenic studies have indicated the involvement of hexokinase (HXK) as a sugar sensor with both signaling and metabolic functions in plants (Jang et al., 1997; Smeekens, 2000; Rolland et al., 2002). To further elucidate the molecular mechanisms underlying the plant sugar signaling network, a genetic approach was used to isolate sugar response mutants in Arabidopsis. Based on a bioassay in which a high level of Glc blocks the switch to postgermination development in Arabidopsis, both Glc-insensitive (gin) and Glc-oversensitive (glo) mutants were isolated. Phenotypic, genetic, and molecular characterization of these mutants has provided new insights into the regulatory mechanisms that link nutrient status to plant hormone synthesis and signaling (Zhou et al., 1998; Arenas-Huertero et al., 2000; Rolland et al., 2002).

Phenotypic analysis of the gin1 mutant has suggested an interaction between the signaling pathways mediated by Glc and the plant stress hormone ethylene. Further characterization of the ethylene overproduction mutant eto1, the constitutive ethylene signaling mutant ctr1, and the ethylene-insensitive mutant etr1 has led to the discovery that the Glc and ethylene signaling pathways antagonize each other and that GIN1 acts downstream of the Glc sensor HXK1 and the ethylene receptor ETR1 (Zhou et al., 1998). Studies of the gin1 (allelic to aba2, sañ3, sis4, isi4, and sre1), gin5 (allelic to aba3 and los5), and gin6 (allelic to abi4, sun6, sañ5, sis5, and isi3) mutants have also revealed that another plant stress hormone, abscisic acid (ABA), is important for Glc responses (Leon-Kloosterziel et al., 1996; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Quesada et al., 2000; Rook et al., 2001; González-Guzmán et al., 2002). Interestingly, recent genetic and phenotypic analyses have revealed complex interactions between ABA and ethyl-ene based on seedling assays (Beaudoin et al., 2000; Ghassemian et al., 2000). Both ABA and ethylene are well-known plant stress hormones with growth-inhibiting activities. How they exhibit opposite roles in Glc responses, and the molecular mechanisms underlying the Glc regulation of ethylene signaling and ABA biosynthesis, remain mostly unclear (Zhou et al., 1998; Arenas-Huertero et al., 2000; Finkelstein and Gibson, 2001; Gazzarrini and McCourt, 2001).

To elucidate the molecular mechanisms underlying the Glc regulation of ABA biosynthesis and ethylene signaling, molecular cloning of ABA2/GIN1 was performed. ABA2/GIN1 encodes the first described member of a large (56-member) short-chain dehydrogenase/reductase (SDR) gene family in Arabidopsis and plays a unique and specific role in the ABA biosynthesis pathway. We show that Glc activates ABA2/GIN1 expression, which is required for antagonizing ethylene signaling. This result also prompted us to examine Glc regulation of the other ABA biosynthesis and signaling genes. These studies provide direct molecular evidence for extensive Glc control of the genes involved in ABA biosynthesis and signaling. Gene expression analyses in the aba2/gin1 mutant also reveal that Glc acts synergistically with endogenous ABA and that Glc regulation is distinct from regulation by osmotic stress.

In addition to a role in Glc signaling, ABA has long been known as a plant hormone important for drought tolerance, by mediating guard cell signaling and stomatal closure to prevent extensive water loss, and for seed maturation, desiccation, dormancy, and germination (Zeevaart and Creelman, 1988; Finkelstein and Zeevaart, 1994; Leung and Giraudat, 1998; Koornneef et al., 2001; Schroeder et al., 2001; Finkelstein et al., 2002). In general, ABA is considered a stress hormone and a growth inhibitor. However, based on examination of the phenotypes of multiple null mutations in ABA2/GIN1 specific to the ABA biosynthesis pathway and transgenic plants complemented with ABA2/GIN1, it is clear that endogenous ABA also plays crucial roles as a growth-promoting hormone in controlling fertility and vegetative growth and determining organ and body size. Thus, ABA possesses dual functions as a growth inhibitor and a growth promoter in the presence and absence of stresses, respectively.

Despite extensive analyses of ABA signaling at target sites, such as guard cells, mesophyll cells, maturing seeds, and germinating seedlings, the precise locations of ABA biosynthesis remain elusive. The SDR-related activities in ABA biosynthesis have been presumed to be constitutive and ubiquitous (Sindhu and Walton, 1987; Schwartz et al., 1997a). Examination of the spatial and temporal expression patterns of an ABA2/GIN1 reporter gene has revealed surprising evidence for its highly restricted localization in specific vascular tissues. These results indicate interorgan and intercellular mobilization of ABA precursors and/or ABA into the target sites. Cellular and biochemical analyses demonstrate conclusively that the multistep conversion of the plastid-derived xanthoxin to abscisic aldehyde (ABAld) is catalyzed by the sole cytosolic SDR1 and is essential for the major and physiological pathway in plant ABA biosynthesis.

RESULTS

Interplays of Glc, ABA, and Ethylene Signaling

Genetic and phenotypic analyses of the Arabidopsis Glc, ethylene, and ABA mutants have suggested a connection between these signaling pathways based on a Glc bioassay in which a high level of Glc (6%) blocks the switch to postgermination development (Zhou et al., 1998; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001). Because three gin1 mutants have a wilty phenotype and the mutations are mapped closely to the ABA-deficient locus aba2 that also displays Glc insensitivity (Figure 1A), reciprocal crosses were performed between gin1-3 and aba2-1. The progeny of both crosses showed Glc insensitivity (Figure 1B), establishing that gin1 and aba2 are allelic. To determine whether ABA deficiency is the cause of the gin1 phenotype in the Glc-dependent bioassay, a noninhibitory level of ABA (100 nM) was added to the Glc medium. At 4% Glc, the addition of ABA restored the Glc sensitivity of gin1 and another ABA-deficient mutant, gin5, but not that of the ABA-insensitive mutant abi4, which is allelic to gin6 (Figure 1C). Although 4% mannitol reduced seedling development in the presence of ABA, the effects of Glc and mannitol were distinguishable (Figures 1C and 1D).

Figure 1.
Genetic and Phenotypic Analyses of Arabidopsis gin1 and gin4 Mutants.

We have shown previously that the constitutive ethylene signaling mutant ctr1 displays a Glc-insensitive phenotype similar to that of gin1 (Zhou et al., 1998). However, after prolonged Glc treatment, ctr1 remained smaller, with epinasty and light green leaves, conspicuous mid vein, and short roots (Figures 1A and 1E), suggesting a partial overlap of the gin and ctr phenotypes. To determine whether any other gin mutants are allelic to ctr1, we examined gin2, gin3, gin4, and gin5 mutants on 6% Glc. Only gin4-1 and gin4-2 mutants resembled ctr1 (Figure 1E); these were crossed to ctr1 and analyzed for the constitutive triple response in etiolated seedlings grown in the dark (Stepanova and Ecker, 2000). The results showed that gin4 is allelic to ctr1 (Figure 1F), confirming an antagonistic interaction between the Glc and ethylene signaling pathways (Zhou et al., 1998). Consistently, another sugar mutant screen also isolated a new ctr1 allele as sis1 (Gibson et al., 2001).

Map-Based Cloning of ABA2/GIN1

To understand the molecular basis and regulation of the ABA2/GIN1 gene, a map-based cloning approach was used. We used PCR and available simple sequence length polymorphism (SSLP) markers (Bell and Ecker, 1994) to localize GIN1 on chromosome 1 near the SSLP markers nga128 and nga280 (Zhou et al., 1998). For fine mapping, we generated 10 new SSLP markers for ecotypes Wassilewskija (Ws), Columbia (Col), and Landsberg erecta (Ler) spanning >1700 kb covered by BAC clones (Figure 2A). After PCR analysis of 5627 DNA samples, the GIN1 gene was localized to a region between BAC clones F11M15 and F6D8 (Figure 2A). One of the genes in the F19K6 BAC clone encodes a putative SDR (SDR1) that might perform the final oxidation steps in converting xanthoxin to ABA (Sindhu and Walton, 1987; Schwartz et al., 1997a) and was the best candidate for ABA2/GIN1. To determine whether there was any molecular defect in the candidate gene transcript in the gin1 mutants, reverse transcriptase PCR (RT-PCR) was performed using primers in the predicted coding sequence of exon 2. Neither gin1-1 nor gin1-3 yielded the expected 762-bp RT-PCR product observed in the wild-type Ws and Col controls, whereas a similar RT-PCR product of the Suc synthase gene (SUS) encoded by the same BAC sequence was detected in both the wild type and the gin1 mutants (Figure 2B). Map-based cloning using the aba2-2 mutant allele also identified the same SDR1 candidate gene (data not shown).

Figure 2.
Map-Based Cloning of ABA2/GIN1.

The full-length cDNA sequence (1179 bp) of ABA2/GIN1 was obtained by 5′ and 3′ rapid amplification of cDNA ends (Figures 2C and 2D) and confirmed by RNA gel blot analysis (see Figure 6A). The deduced polypeptide is predicted to be 285 amino acids in length without an overt transmembrane domain (Figures 2D and 2E). To further validate the notion that ABA2/GIN1 encodes the putative SDR1, the molecular bases of seven aba2/gin1 alleles (aba2-1, aba2-2, aba2-3, aba2-4, gin1-1, gin1-2, and gin1-3) were analyzed and summarized (Figure 2E). At least three alleles, gin1-1, gin1-3, and aba2-2, appeared to be null mutations producing defective or no transcript (Figures 2B, ,2E,2E, and and6A).6A). The gin1-3 allele has a 53-bp deletion in exon 2, causing a frameshift after 22 amino acids and an early translation stop after 32 amino acids (Figure 2E). We observed no protein production with a gin1-3 and green fluorescent protein (GFP) fusion (data not shown). Both gin1-1 and aba2-2 have variable intron mutations that gave no RT-PCR product for gin1-1 (Figures 2B and 2E) and no aba2-2 transcript (see Figure 6A) when analyzed with 5′-end exon 2 sequences. Five additional aba2 alleles also have been analyzed independently by other groups (Figure 2E) (Laby et al., 2000; Rook et al., 2001; González-Guzmán et al., 2002).

Figure 6.
Tissue, Stress, and ABA Regulation of Gene Expression.

ABA2/GIN1 Encodes a Unique Member of the SDR Superfamily

Based on a BLASTP search, ABA2/GIN1 is a member of a large SDR gene family. All 56 of the putative Arabidopsis SDR members range between 250 and 300 amino acids in length and have a NAD/NADH binding motif (TGXXXGXG) and a catalytic site (YXXS/T/AK) that are characteristic of and conserved in SDR superfamily members in Escherichia coli, yeast, flies, worms, mice, humans, and plants (Figure 3) (Jörnvall et al., 1999; Oppermann et al., 2001). The ABA2/GIN1 gene encoding Arabidopsis SDR1 is likely to have a unique function, because the most closely related member, SDR2 (with 62% similarity to SDR1), is a homolog of the maize TASSELSEED2 (ZmTS2, with 73% similarity to AtSDR2) that is important for sex determination (Figure 3) (DeLong et al., 1993). Judging from the severe phenotypes displayed in the null alleles of aba2/gin1 mutants and the homology of AtSDR2 with ZmTS2, Arabidopsis SDR1 has nonredundant functions.

Figure 3.
Arabidopsis ABA2/GIN1 Encodes a Unique Member of the SDR Superfamily.

ABA2/GIN1 as a Link between Glc and Ethylene Signaling

To determine the link between ethylene and ABA pathways in Glc responses, a double mutant of the strongest ethylene-insensitive mutant, ein2 (Alonso et al., 1999), displaying Glc oversensitivity (Figure 4A), and the Glc-insensitive mutant gin1 was generated. Because the Glc-oversensitive phenotype of ein2 on 4% Glc was blocked in the ein2 gin1 double mutant (Figure 4A), ethylene could contribute to the Glc-dependent phenotypes by antagonizing ABA biosynthesis, as suggested previously (Ghassemian et al., 2000). To determine whether ethylene signaling is antagonized by Glc through ABA, the expression of an ethylene-regulated gene, PDF1.2 (Alonso et al., 1999), was examined. The presence of 2 and 6% Glc repressed PDF1.2 expression that requires endogenous ethylene in the wild type but not in aba2/gin1 (Figure 4B). Unlike 2% Glc, 2% mannitol as an osmotic control did not repress PDF1.2 expression in either the wild type or aba2/gin1. Unlike 6% mannitol, 6% Glc strongly repressed PDF1.2 expression in the wild type but activated its expression in aba2/gin1 (Figure 4B). These results support the model that the antagonistic relationship between Glc and ethylene signaling is mediated in part through ABA2/GIN1 involvement in ABA biosynthesis. Although there is an ABA-dependent osmotic effect at 6% mannitol, the Glc and mannitol signals are distinguishable.

Figure 4.
Interplay of Glc, ABA, and Ethylene Signaling Pathways.

ABA as a Growth-Promoting Regulator

Although ABA has long been considered a stress hormone that accumulates under stress conditions and a growth inhibitor when applied exogenously, the ABA-deficient mutant aba2/gin1 exhibited severe growth retardation in cotyledons, rosettes, stems, roots, and siliques in the absence of exogenous sugars and stress conditions (Figures 4C and 4D). Thus, endogenous ABA also could be critical for the promotion of plant growth. This dual function of ABA is similar to that of Glc in promoting growth at low concentrations but inhibiting growth at high concentrations (Xiao et al., 2000). Examined with a dissecting microscope, it appeared that the smaller cotyledons were attributable to a lack of cell expansion and the smaller rosettes were attributable to a reduction in both cell number and size (data not shown). The root system of aba2/gin1 was shorter, with fewer lateral roots (Figures 4C and 4D). A similar growth defect was found when aba2/gin1 plants were grown under high humidity (Figure 4D). To further test the idea that the growth defect of aba2/gin1 is not caused by chronic water stress, wild-type and aba2/gin1 plants were grown in a container with nearly 100% RH. As shown in Figure 4E, the rosettes and root system of aba2/gin1 remained significantly smaller, even though the leaf expansion of the wild type also was limited under this condition. The aba2/gin1 mutant showed a wilty phenotype within 5 min of exposure to lower humidity (Figure 4E), supporting the importance of basal ABA levels in the guard cell response to water stress. It has been suggested that the growth defect in tomato ABA-deficient mutants is the result of an inhibitory effect of ethylene (Sharp et al., 2000). By comparing the phenotypes of aba2/gin1, ein2, and gin1 ein2 plants, it was obvious that the growth retardation of aba2/gin1 shoots could be attributed to ABA deficiency even in the completely ethylene-insensitive ein2 background (Alonso et al., 1999), regardless of the humidity (Figure 4F) (data not shown). However, the root phenotype of the gin1 ein2 double mutant resembled that of ein2 but not that of aba2/gin1 (Figure 4F). These results support the antagonistic roles of ABA and ethylene in root development and a surprising uncoupling of the shoot and root phenotypes modulated by endogenous ABA.

Complementation

To ensure that ABA2/GIN1 encodes SDR1 and that the aba2/gin1 phenotypes are the consequence of a single gene mutation, complementation was performed by transforming the null gin1-3 mutant with a 7.7-kb ABA2/GIN1 genomic clone that includes the 3-kb promoter or with the full-length cDNA under the control of a constitutive 35S promoter (35S::GIN1) in a miniature binary vector (pCB302) through Agrobacterium tumefaciens (Xiang et al., 1999). Ten independent lines with clear complementation in the greenhouse were brought to homozygous T3 generation before detailed characterization. The ABA2/GIN1 genomic clone and 35S:: GIN1 restored the Glc sensitivity of gin1 (Figure 5A and data not shown). The growth defects in cotyledons and rosettes (Figure 5B), lateral roots (Figure 5C), stems (Figure 5D), siliques (Figure 5E), and embryos (Figure 5F), as well as the wilty (Figure 5G) and lack of seed dormancy (Figure 5H) phenotypes of gin1, also were complemented. These results are consistent with the dual function of endogenous ABA as a growth-promoting regulator in addition to its roles in Glc signaling, stress tolerance, and seed dormancy.

Figure 5.
Complementation.

Tissue, Stress, and ABA Regulation of ABA Biosynthesis Gene Expression

To determine the tissue-specific expression patterns of ABA2/GIN1, RNA gel blot analysis was performed. Surprisingly, ABA2/GIN1 was expressed predominantly in roots and stems but at much lower levels in rosettes and seeds, the established ABA target sites (xA). Analyses of the petiole and blade RNA samples isolated from “rosettes” further supported the preferential expression of ABA2/GIN1 outside of the presumed ABA target sites, mesophyll, and guard cells. Unless the ABA2/GIN1 protein is mobile, interorgan and intercellular transport of ABA precursors and/or ABA likely occurs to coordinate ABA biosynthesis and response, because genes that encode other ABA biosynthesis enzymes, such as zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED3), and Arabidopsis aldehyde oxidase 3 (AAO3), are expressed differentially (Seo et al., 2000a; Xiong et al., 2002; M. Seo and T. Koshiba, unpublished data). Interestingly, Commelina stomatal closure is induced by xanthoxin when it is added through the transpiration stream but not by direct application on epidermal strips (Raschke et al., 1975).

It has been shown that genes important for ABA biosynthesis, such as Arabidopsis ZEP (ABA1/LOS6), NCED3, AAO3, and ABA3 (LOS5), are induced by water stress (Qin and Zeevaart, 1999; Seo et al., 2000a; Iuchi et al., 2001; Xiong et al., 2001, 2002), although the enzymes that convert xanthoxin to ABAld and ABA are detected constitutively in leaves (Sindhu and Walton, 1987). In contrast to the NCED3 transcript that was induced rapidly by dehydration and absent in water, the ABA2/GIN1 transcript did not increase during dehydration treatment (Figure 6B). Thus, ABA2/GIN1 is important for the basal level of ABA but is not the rate-limiting factor for the stress-inducible accumulation of ABA (Qin and Zeevaart, 1999; Iuchi et al., 2000). The expression of ZEP (ABA1/LOS6), NCED3, and AAO3 also was induced by ABA in shoots and roots, but the expression of ABA2/GIN1 was not (Figure 6C). It appears that the expression of ABA2/GIN1 is not coordinated with that of other ABA biosynthesis genes by stress and ABA.

Glc Regulation of ABA Biosynthesis and Signaling Gene Expression

To determine how Glc controls ABA accumulation, the expression of ABA2/GIN1 was examined with or without exogenous Glc in wild-type and aba2/gin1 seedlings. As shown in Figure 7, the ABA2/GIN1 transcript was induced significantly by 2 and 6% Glc only in wild-type but not in aba2/gin1 seedlings. Because ABA2/GIN1 is not induced by ABA (Figure 6C) but ABA deficiency in aba2/gin1 prevented its Glc induction, it is likely that both Glc and ABA are required synergistically for ABA2/GIN1 regulation. Mannitol at 2 and 6% also induced ABA2/GIN1 expression approximately twofold in an ABA-dependent manner (Figure 7). To investigate the extent of Glc regulation, we also examined other genes involved in ABA biosynthesis and ABA signaling by RT-PCR. The results showed that other ABA biosynthesis genes, such as ABA1, AAO3, and ABA3, were upregulated by 2 and 6% Glc, similar to the effect found for ABA2/GIN1 (Figure 7). Glc activation was abolished in the gin1-3 null mutant, suggesting a critical role of endogenous ABA in the Glc induction of ABA biosynthesis genes (Figure 7). Interestingly, unlike 6% Glc, which activated gene expression, 6% mannitol repressed these genes in both the wild type and aba2/gin1 (Figure 7). The expression of NCED3 was extremely low and not activated by Glc, distinguishing Glc signals from stress signals that activated NCED3 (Figures 6B and 6C). Interestingly, ABI3, ABI4, and ABI5 but not ABI1 and ABI2 transcripts showed dramatic induction by 6% Glc but not by 6% mannitol. The induction was eliminated completely in aba2/gin1 (Figure 7). These results are consistent with the Glc-insensitive phenotypes shown in the aba1, aba2, aba3, abi4, and abi5 mutants (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000). As controls, the expression of a Glc sensor encoded by AtHXK1 and a ubiquitin gene (UBQ5) was not influenced by Glc or mannitol (Figure 7) (Jang et al., 1997).

Figure 7.
Glc Regulation of Gene Expression.

Restricted Spatial and Temporal Expression of ABA2/GIN1

To determine the spatial and temporal expression patterns of ABA2/GIN1 more precisely, the ABA2/GIN1 promoter (2906 bp upstream of the ATG start codon) was fused to a reporter gene encoding β-glucuronidase (GUS) to generate GIN1::GUS. GUS was chosen for its high sensitivity because ABA2/GIN1 is not highly expressed. The promoter was used successfully for complementation in the 8-kb genomic clone. The GIN1::GUS fusion was introduced into the wild type and the gin1-3 null mutant through Agrobacterium-mediated transformation (Jang et al., 2000). In general, GIN1:: GUS had similar expression patterns in multiple independent transgenic lines of both the wild-type and gin1 backgrounds under normal growth conditions, based on staining with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid. However, GUS staining usually was stronger in aba2/gin1 than in the wild type, suggesting negative feedback control by ABA in the absence of high exogenous Glc (see below). GUS staining was readily visible in vascular bundles of hypocotyls (Figures 8A and 8B) and the branching points of lateral and primary roots (Figure 8C) of wild-type and aba2/gin1 transgenic seedlings grown on soil for 10 days after germination. GUS staining in hypocotyls became weak and started to appear in petioles of cotyledons and true leaves at 20 days (Figures 8D and 8E). The staining signal in petioles of cotyledons and the first pair of true leaves became more pronounced at 30 days, especially in the aba2/gin1 background (Figure 8F). The staining at branching points of primary and lateral roots remained visible at maturity.

Figure 8.
Spatial and Temporal Expression Patterns of ABA2/GIN1.

In adult plants (after 30 days), pollen showed strong GUS staining in opening and opened flowers but not in flower buds and unopened flowers (Figure 8G). There was strong GUS staining at the junctions of pedicels and young developing siliques (Figure 8H) but not before petal, anther, and sepal abscission (Figure 8G), suggesting a role of ABA in early embryogenesis. At a later stage (57 days), GUS staining appeared in the vascular bundles of petioles and the mid veins of aged rosette leaves, stems, and the junctions of pedicels and siliques (data not shown). GUS staining was strong in septa and funiculi inside maturing siliques but was faint in seeds (Figure 8I) and developing embryos (data not shown). These GUS staining results are consistent with RNA gel blot analyses of ABA2/GIN1 expression in specific tissues (Figure 6A). It appears that ABA2/GIN1 has restricted expression patterns in hypocotyls, petioles, leaf veins, stems, flowers, pedicels, septa, funiculi, and roots but is not abundant in the ABA target sites, such as mesophyll cells, guard cells, and seeds.

Distinct Glc Regulation of the ABA2/GIN1 Promoter

Although the expression of ABA2/GIN1 does not seem to be affected significantly by drought or ABA, based on RNA gel blot analysis in the wild type, we examined GUS staining to determine whether the expression pattern changes as a result of salt stress (250 mM NaCl for 6 h) and cold stress (4°C for 6 h) in the wild type and aba2/gin1. For salt or cold treatment of 10-day-old seedlings, GUS staining showed no detectable changes in the wild type (Figures 8A, 8B, 8J, and 8L). However, GUS staining showed a significant increase in vascular bundles of cotyledons in aba2/gin1 (Figures 8K and 8M). Because ABA2/GIN1 showed a slight downregulation by ABA in RNA gel blot analysis (Figure 6C), the enhanced GUS staining may reflect the lack of this negative ABA feedback control in aba2/gin1.

Because ABA2/GIN1 expression was enhanced by Glc (Figure 7), we examined the GUS expression patterns in both the wild type and aba2/gin1 in the presence of 2 and 6% Glc. Consistent with the results of RNA gel blot analysis (Figure 7), the presence of 2% Glc or mannitol enhanced GUS expression significantly, which is restricted to the vascular bundles of cotyledons in addition to hypocotyls and roots (Figures 8N, 8O, 8Q, and 8R). Although the aba2/gin1 mutant transcript was not enhanced by 2% Glc (Figure 7), the ABA2/GIN1 promoter still was activated by Glc in gin1 (Figure 8O). This result suggests a direct regulation of the ABA2/GIN1 promoter by Glc independent of ABA. The stability of the ABA2/GIN1 transcript may be ABA dependent. Importantly, 6% Glc activated the ABA2/GIN1 promoter more dramatically and caused developmental arrest in the wild type (Figure 8P). Both Glc and ABA were required for the effect of 6% Glc, because the intense GUS staining and developmental arrest were not observed in aba2/gin1 (Figure 8P). Treatment with 6% mannitol to induce osmotic stress did not mimic the effect of 6% Glc in the wild type and aba2/gin1. However, 6% mannitol did result in more intense and broader GUS staining in the whole seedlings, including the root tips (Figures 8S and 8T). These results are consistent with the idea that Glc and mannitol exert distinguishable regulatory mechanisms in ABA biosynthesis and seedling development. A quantitative GUS assay further supported the distinct responses of the ABA2/GIN1 promoter to 6% Glc and 6% mannitol (Figure 9). There appear to be multiple mechanisms for the regulation of the ABA2/GIN1 promoter and ABA2/GIN1 transcripts (Figures 6 to to9).9). The dynamic regulation of ABA2/GIN1 and the distinct 6% Glc regulation of other ABA biosynthesis and signaling genes (Figure 7) seem to be important for the Glc-dependent developmental arrest (Figure 8P).

Figure 9.
Glc Regulation of ABA2/GIN1 Promoter Activity.

Subcellular Localization and Biochemical Analyses of ABA2/GIN1

It has been suggested that the complete ABA biosynthesis pathway could exist inside chloroplasts (Milborrow, 2001). However, biochemical analyses of ABA biosynthetic enzymes have indicated that the last steps of ABA biosynthesis may occur in the cytosol (Sindhu and Walton, 1987). To determine the subcellular localization of the ABA2/GIN1 protein in Arabidopsis, a GFP fusion protein, GIN1-GFP, was generated. Using the Arabidopsis protoplast transient expression assay and confocal microscopy, we showed that GIN1-GFP was localized exclusively in the cytosol, similar to a cytosolic GFP marker used as a control (Figure 10A) (Hwang and Sheen, 2001).

Figure 10.
ABA2/GIN1 Subcellular Localization and Biochemical Activities.

To determine whether the Glc-insensitive phenotype of aba2/gin1 is attributable to a deficiency in the Glc induction of ABA accumulation, the endogenous levels of ABA were measured in wild-type, aba2/gin1, and transgenic seedlings complemented with the ABA2/GIN1 genomic clone. As shown in Figure 10B, aba2/gin1 seedlings had a low basal ABA level that was not induced by Glc, as in the wild type. The ABA2/GIN1 genomic clone complemented the basal and Glc-induced levels of ABA in transgenic plants (Figure 10B), supporting a critical role of ABA2/GIN1 in endogenous ABA biosynthesis and in Glc responses. The growth-promoting and growth-inhibiting dual functions of ABA may have different threshold levels.

Although it has been suggested that xanthoxin is converted to ABAld by SDR1 and then to ABA by AAO3 (Sindhu and Walton, 1987; Zeevaart and Creelman, 1988; Schwartz et al., 1997a; Seo et al., 2000a, 2000b), the possibility remained that xanthoxic acid but not xanthoxin is the substrate for SDR1 (Cowan, 2000; Seo and Koshiba, 2002). Moreover, because three chemical reactions are required to convert xanthoxin to ABAld, it is unclear whether enzymes other than SDR1 might be required (Cowan, 2000; González-Guzmán et al., 2002; Seo and Koshiba, 2002). To determine the SDR1 substrate specificity and whether SDR1 alone can catalyze multiple chemical reactions, recombinant SDR1 was expressed in the yeast Pichia pastoris for in vitro enzymatic analyses (Koiwai et al., 2000). His-tagged SDR1 was purified to homogeneity using ammonium sulfate fractionation, Ni2+ column chromatography, and DEAE column chromatography and analyzed by SDS-PAGE, as shown in Figure 10C. The purified recombinant SDR1 catalyzed xanthoxin (Km of 20.2 μM) but not xanthoxic acid as a substrate and generated a product with the same retention time as that of ABAld, as detected by HPLC and gas chromatography–mass spectrometry with a mother fragment with a mass-to-charge ratio of 246 (Figure 10D). The reaction absolutely required a coenzyme NAD (data not shown). Consistent with these results, AAO3 did not use xanthoxin as a substrate, as determined by HPLC (data not shown). Thus, our data provide conclusive evidence that the final steps of the major endogenous ABA biosynthesis pathway involve two enzymes, SDR1 and AAO3, which convert xanthoxin to ABAld and then convert ABAld to ABA, respectively.

DISCUSSION

ABA in Glc Signaling

Genetic and phenotypic analyses of the Arabidopsis mutants displaying gin and glo phenotypes have revealed extensive interactions between the metabolic and hormonal signaling pathways (Zhou et al., 1998; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Gibson et al., 2001; Rook et al., 2001). Our studies provide molecular evidence for direct and extensive Glc control of the genes important for ABA biosynthesis and signaling. Interestingly, in the case of ABA2/GIN1, ABI3, and ABI4, Glc and ABA act synergistically. These genes are not activated by ABA alone, but their Glc activation requires endogenous ABA and is abolished in the aba2/gin1 mutant (Figures 6 and and7)7) (Arenas-Huertero et al., 2000; Söderman et al., 2000). In most cases, plants display different responses to Glc and mannitol signals. Thus, our studies show that Glc signaling is mediated partly by the activation of ABA biosynthesis and signaling genes through two mechanisms—a synergistic effect of Glc and ABA, and an ABA positive feedback control loop—because ABA1/ZEP, AAO3, ABA3, and ABI5 also are activated by ABA (Figures 6 and and7)7) (Leung et al., 1997; Lopez-Molina et al., 2001; Xiong et al., 2001, 2002). The synergistic interaction between Glc and ABA also controls seedling development. Increased ABA accumulation stimulated by a high level of Glc (Figure 10B) is required to block the postgermination developmental switch, demonstrating ABA's function as a growth inhibitor. However, this Glc induction of ABA levels and ABA biosynthesis gene expression is not the result of differences in developmental stage and morphology in seedlings. As shown previously, Glc induction of ABA levels occurs in gin6/abi4 without a developmental arrest phenotype (Arenas-Huertero et al., 2000).

Our studies also reveal that Glc antagonizes the responses of endogenous ethylene through ABA. For example, the Glc hypersensitive phenotypes of ein2 and the Glc repression of an ethylene maker gene, PDF1.2, are eliminated in the aba2/gin1 mutant. In this condition, ethylene acts as a growth-promoting regulator (Zhou et al., 1998), as has been shown in hypocotyl elongation under light (Smalle et al., 1997; Alonso et al., 1999). In the absence of a high level of Glc and at basal ABA levels, the physiological roles of endogenous ABA and ethylene are reversed. In addition, ethylene also may repress ABA biosynthesis, as observed in the ein2 mutant (Ghassemian et al., 2000), or ABA signaling (Beaudoin et al., 2000). Thus, Glc signals can profoundly influence plant hormone biosynthesis and actions and plant growth and development (Figure 11).

Figure 11.
Model of the Interplay of the Glc, ABA, and Ethylene Signaling Pathways.

Distinct Glc and Osmotic Signaling

The expression of NCED is highly activated by stress conditions and encodes a rate-limiting enzyme for the stress-inducible accumulation of ABA in many plant species (Tan et al., 1997; Qin and Zeevaart, 1999; Iuchi et al., 2000, 2001). Here, we show that Glc does not activate NCED3 significantly in Arabidopsis. This finding suggests that stress- and Glc-induced ABA accumulation is mediated by distinct mechanisms. This notion is consistent with the differences in seedling morphology and GIN1::GUS expression patterns after 6% Glc and 6% mannitol treatments. aba2/gin1 mutant alleles have been obtained from a variety of other screens for Suc mutants (sis and isi) (Laby et al., 2000; Rook et al., 2001) and salt resistance mutants (sañ and sre) (Quesada et al., 2000; González-Guzmán et al., 2002). The common feature of these mutations is the important role of ABA in sugar and salt/osmotic signaling, although Glc and stress signaling are distinct in their ABA regulation.

For the Glc bioassay under which gin and glo mutants were screened, the imbibition phase and the radicle protrusion of Arabidopsis seeds (Bewley, 1997; Koornneef et al., 2001) are not blocked by Glc (Jang et al., 1997; Zhou et al., 1998). However, excess Glc signals block the switch to postgermination development, including cotyledon greening and expansion, as well as apical shoot meristem and root differentiation (Jang et al., 1997; Zhou et al., 1998). This developmental arrest imposed by Glc presumably signals a feedback regulation in the source tissues, the seeds that provide carbon nutrients for seedling growth. Under natural conditions, an inappropriate environment could limit early seedling growth and sugar consumption after germination, resulting in Glc accumulation and developmental arrest. This safeguard process for early seedling development involves newly synthesized and increased ABA (Figure 10B) and transcription factors ABI3, ABI4, and ABI5 controlled by Glc (Figure 7). Glc regulation differs from osmotic, salt, and drought stress controls (Leon-Kloosterziel et al., 1996; Quesada et al., 2000; González-Guzmán et al., 2002) by modulating additional signaling processes of other plant hormones, including ethylene, auxin, and cytokinin (Zhou et al., 1998; Sheen et al., 1999; Rolland et al., 2002; L. Zhou and J. Sheen, unpublished data). Feedback control by Glc is well documented in other types of source tissues, such as photosynthetic leaves, that provide carbon nutrients for the development of sink tissues, including young leaves, roots, stems, flowers, and seeds (Sheen, 1994; Koch, 1996; Stitt and Krapp, 1999; Wobus and Weber, 1999; Smeekens, 2000; Rolland et al., 2002). We propose that endogenous Glc signals could be indicators of the plant growth environment and are monitored in different cell types and organs to adjust source (carbon export) and sink (carbon import) interactions through the regulation of genes involved in plant hormone synthesis and actions and in metabolism, thus coordinating plant growth and development with the environment.

ABA in Growth Promotion

ABA has long been defined as a stress hormone and growth inhibitor because of its roles in stress responses and its inhibitory growth effects when applied exogenously. However, the growth retardation phenotypes of ABA-deficient mutants have implicated an essential function of endogenous ABA in the promotion of leaf and stem development (Koornneef, 1986; Zeevaart and Creelman, 1988; Rock and Zeevaart, 1991; Finkelstein and Zeevaart, 1994; Nambara et al., 1998; Zhou et al., 1998; Sharp et al., 2000). It has been suggested that the pleiotropic phenotypes of ABA-deficient mutants are the result of auxin and ethylene production and/or an indirect effect of photoprotection or water relations (Koornneef, 1986; Sharp et al., 2000). The Arabidopsis ABA-insensitive mutants abi1, abi2, abi3, abi4, and abi5 do not display overt morphological abnormality, probably as a result of tissue or pathway specificity and/or functional redundancy (Finkelstein and Zeevaart, 1994; Rock and Quatrano, 1995; Leung and Giraudat, 1998; Söderman et al., 2000). Analyses of Arabidopsis mutants in the ABA biosynthesis pathway, such as aba1, aba3, aao3, and the NCED3 knockout mutant, have not offered a solution because the ABA1 and ABA3 gene products have functions other than ABA biosynthesis (Koornneef, 1986; Leon-Kloosterziel et al., 1996; Schwartz et al., 1997a), whereas the aao3 mutant and a NCED3 knockout mutant lack severe morphological phenotypes, presumably as a result of functional redundancy provided by other members of the multigene families (Seo et al., 2000a; Iuchi et al., 2001; Seo and Koshiba, 2002). Molecular cloning and BLAST searches have revealed that ABA2/GIN1 encodes a unique SDR1 specific to the ABA biosynthesis pathway. At present, the aba2 mutants are available only in Arabidopsis (Seo and Koshiba, 2002). Thus, analyses of the Arabidopsis aba2/gin1 null mutants and complementation by ABA2/GIN1 in transgenic plants provide an opportunity to reevaluate the physiological functions of ABA in growth promotion. It is clear that endogenous ABA is important for cotyledon, leaf, root, stem, and silique development and fertility (Figures 4, ,5,5, and and12).12). Blocking ethylene signaling partially restores root growth and development in aba2/gin1, but other phenotypes are independent of ethylene, even when grown under high-humidity conditions.

Figure 12.
Proposed ABA Biosynthesis Pathway and Functions.

The size and morphology of flowers and mature seeds in aba2/gin1 are similar to those in the wild type (data not shown). However, the smaller aba2/gin1 siliques have many unfertilized ovaries and/or aborted embryos at early stages and produce many fewer mature seeds than do wild-type siliques (Figures 5E and 5F). ABA may play a role in the early embryo differentiation found during the study of somatic embryogenesis (Zimmerman, 1993; Rock and Quatrano, 1995). The striking expression of GIN1::GUS at the junction of the pedicel and the young silique is consistent with such a role (Figure 8H). The precise stage and mechanisms of ABA function during early embryogenesis require further investigation. The expression of GIN1::GUS also is prominent in the septa and funiculi of mature siliques (Figure 8I), suggesting a maternal supply of ABA for seed maturation, dormancy, and/or desiccation (Figure 12), as shown by genetic analysis in Arabidopsis (Raz et al., 2001).

Analysis of the aba2/gin1 mutants also supports the idea that preexisting ABA is critical to rapid guard cell signaling (within minutes) in response to water stress (Figure 12). Stress-induced ABA accumulation requires de novo transcription and translation of NCED as a rate-limiting enzyme and takes hours to occur (Qin and Zeevaart, 1999; Iuchi et al., 2000). Overexpression of NCED3 in transgenic plants enhances drought tolerance over that of the wild type (Iuchi et al., 2001; Qin and Zeevaart, 2002), indicating that high ABA levels may be important for long-term survival under severe growth conditions. Although not inducible under stress conditions, ABA2/GIN1 plays a key role in the support of many physiological functions of ABA that do not require induction by stress. There could be a physiological connection between Glc and ABA at lower endogenous levels that promotes rather than inhibits plant growth.

ABA Biosynthesis Pathway and Regulation

ABA is a sesquiterpenoid (C15) and is produced from an indirect pathway from the cleavage products of carotenoids (C40) in the plastids (Figure 12) (Zeevaart and Creelman, 1988; Seo and Koshiba, 2002). The cloning and biochemical analysis of the first ABA pathway–specific enzyme, NCED, encoded by maize VP14, provided conclusive evidence that the ABA precursor xanthoxin is generated from the cleavage of 9-cis-epoxycarotenoids by NCED (Schwartz et al., 1997b; Tan et al., 1997). Cellular expression and chloroplast import studies show that NCED is localized in the chloroplasts (Figure 12) (Qin and Zeevaart, 1999; Iuchi et al., 2000; Tan et al., 2001). Recent genetic and biochemical characterizations of the Arabidopsis aba2, aao3, and aba3 mutants and the tomato sitiens and flacca mutants have supported the conversion of xanthoxin to ABAld and ABA as the final steps (Figure 12) (Leon-Kloosterziel et al., 1996; Schwartz et al., 1997a; Sagi et al., 1999; Seo et al., 2000a, 2000b; Seo and Koshiba, 2002). However, the location at which, and the chemical reactions by which, xanthoxin is converted to ABA remain controversial. For example, the final steps of ABA biosynthesis have been observed to occur in both chloroplasts and cytosol (Sindhu and Walton, 1987; Milborrow, 2001). Abscisic alcohol and xanthoxic acid, instead of ABAld, have been proposed to be metabolic intermediates (Cutler and Krochko, 1999; Cowan, 2000; Milborrow, 2001; Seo and Koshiba, 2002). Our cellular analysis of GIN1-GFP localization and biochemical analyses of the purified enzyme support the conversion of xanthoxin to ABAld by SDR1 in the cytosol in plants (Figure 12). This physiological function of SDR1 is further confirmed by the fact that SDR1 cannot use xanthoxic acid as a substrate and AAO3 can use only ABAld but not xanthoxin as a substrate. However, other minor ABA biosynthesis pathways may exist in aba2/gin1 null mutants, and these require further investigation (Cowan, 2000; Seo and Koshiba, 2002).

Many genes involved in the biosynthesis of plant growth hormones, such as gibberellins, brassinosteroids, and sterols, are expressed in actively growing cells and in the target sites (Silverstone et al., 1997; Jang et al., 2000; Bishop and Yokota, 2001). The enzymes that catalyze the conversion of xanthoxin to ABA were thought previously to be ubiquitous and expressed constitutively (Sindhu and Walton, 1987; Schwartz et al., 1997a; Sagi et al., 1999; Seo et al., 2000a). Unexpectedly, the spatial and temporal expression patterns of ABA2/GIN1 are restricted to specific areas of vascular tissues and are distinct from the tissue expression patterns of other genes, such as ZEP, NCED3, AAO3, and ABA3, that are important for ABA biosynthesis (Audran et al., 1998; Qin and Zeevaart, 1999; Iuchi et al., 2000, 2001; Thompson et al., 2000; Xiong et al., 2001, 2002; M. Seo and T. Koshiba, unpublished data). These results indicate that interorgan, intercellular, and interorganelle transport of ABA precursors and/or ABA may be required for the completion of ABA synthesis and to reach target sites (Figure 12). Additional cellular analyses of promoter fusions and immunolocalization of the ABA biosynthesis enzymes and ABA will be required to understand the physical location and regulation of the ABA biosynthesis pathway.

Physiological Functions of the SDR Superfamily in Eukaryotes

The SDR superfamily represents a very large family of enzymes, most of which are known to be NAD- or NADP-dependent oxidoreductases from E. coli to humans. Because the first characterized member of this family was the novel alcohol dehydrogenase in Drosophila, this family used to be called “insect-type” or “short-chain” alcohol dehydrogenase. The discovery of 15-hydroxyprostaglandin dehydrogenase as the first mammalian SDR brought general interest to SDR functions (Jörnvall, 1999). The first cloned plant SDR gene was maize TS2, which is involved in sex determination by controlling cell death through unknown mechanisms (DeLong et al., 1993). It was suggested, based on its sequence homology with hydroxysteroid dehydrogenases (HSDs) in prokaryotes, that ZmTS2 might control plant steroid metabolism (DeLong et al., 1993). However, sequence alignment revealed that the putative Arabidopsis HSD is evolutionarily distant from ABA2/GIN1/SDR1, SDR2, and ZmTS2 (Figure 3). Although many plant genes involved in brassinosteroid and sterol biosynthesis have been isolated using mutants in Arabidopsis, none encodes SDR (Bishop and Yokota, 2001). The molecular cloning of ABA2/GIN1 reveals the functional significance of plant SDR in ABA biosynthesis: it is critical for growth and development, stress responses, and Glc signaling (Figure 12). The tobacco and cucumber SDR genes are closely related to ABA2/GIN1 and may be involved in ABA biosynthesis (Figure 3). It remains to be determined whether other distinct SDR genes may provide a low level of activity for ABA biosynthesis with different expression patterns. The Arabidopsis TS2 homolog (SDR2) is related more closely to SDR1 than to HSDs of 56 SDR members in the Arabidopsis genome. It will be interesting to determine the functions of Arabidopsis SDR2 and HSD genes by isolating and characterizing knockout mutants and performing enzymatic assays. In mammals, SDRs play diverse and important roles, as in the metabolism of steroid and retinoid hormones and the interaction with amyloid B-peptide in human Alzheimer's disease (Oppermann et al., 1999, 2001). Functional analyses of plant SDRs may provide useful clues to SDR functions in hormone biosynthesis and the metabolic signaling that controls embryogenesis and postnatal growth and development in mammals.

METHODS

Mutant Isolation and Genetic Analyses

Three gin1 mutants, gin1-1 (Wassilewskija [Ws] ecotype), gin1-2 (Landsberg erecta [Ler]), and gin1-3 (Columbia [Col] ecotype), and two gin4 mutants, gin4-1 (Col) and gin4-2 (Col), were selected on 7% Glc Murashige and Skoog (1962) plates as described by Zhou et al. (1998). Allelism tests showed that none of the gin1 or gin4 mutants complemented each other in the Glc bioassay or in the greenhouse. Although selected from T-DNA–tagged pools, none was tagged by T-DNA. The null mutant gin1-1 was used for cloning, and the null mutant gin1-3 was used for all other studies except for those shown in Figure 6A, for which null aba2-2 was used. All mutants were recessive and backcrossed to the wild type to ensure the cosegregation of phenotypes before extensive fine mapping was performed. For the analysis of gin1-1 and gin1-3 reverse transcriptase (RT) PCR products, the following exon 2 primers were used: 5′-AGTGGCATTGATCACTGGAG-3′ and 5′-GTGAATCCTCCATCAATCATC-3′. Note that the 5′ primer sequence was deleted in the gin1-3 mutant except for the initial AGT.

Map-Based Cloning of GIN1

The mapping population was generated by crossing gin1-1 (Ws) to the Col or Ler wild type. F2 populations of gin1-1 mutants (5627) were isolated on 7% Glc Murashige and Skoog (1962) plates. Genomic cDNA was extracted to perform PCR using simple sequence length polymorphism (SSLP) markers to identify recombinants as described previously (Bell and Ecker, 1994). Fine mapping was performed by designing new SSLP markers. The primers were synthesized based on BAC DNA sequences and tested by PCR using DNA isolated from three ecotypes. New SSLP primer sequences and PCR fragment sizes (Col/Ler/Ws, in bp) for fine mapping are listed below: F6D8-33-A3, 5′-ACATGGCTAGAAGGTGGAGAC-3′, and F6D8-33-B3, 5′-ATATTATGCTCCAGTATTTAGG-3′ (183/194/183); F6D8-3-A1, 5′-TCTACATCTAGACGGGAAGG-3′, and F6D8-3-B1, 5′-CGC-CATTGCTTCTCGACTTG-3′ (1140/900/1140); F5F19-19-A3, 5′-ACG-TGTTCACAGCAACATCAAG-3′, and F5F19-19-B3, 5′-GTATTT-TCAGACTAAGCTTTTGCC-3′ (96/93/90); F5F19-12-A1, 5′-CCA-GTTGCTATCCATATCTATTG-3′, and F5F19-12-B1, 5′-ATTCCTCATATGAGATCCGAGG-3′ (180/194/178); F5F19-1-3A, 5′-CTGTGG-TTAGACTGACAAGTG-3′, and F5F19-1-3B, 5′-ACCAAAGAG-CCCCTGAAGAC-3′ (105/105/96); F11M15-24-2A, 5′-AATGGTTAG-ATTCATTAGGATG-3′, and F11M15-24-2B, 5′-TTCTCTGTTTGTGAAGCCAGC-3′ (123/156/123); F11M15-1-A2, 5′-TGGAGTTGC-TGAACCTAGCC-3′, and F11M15-1-B2, 5′-ATGGATCGAAAATAT-GAGTATC-3′ (129/129/120); and F13F21-15-A3, 5′-GTTGAACGG-TTTAGATTGAAGG-3′, and F13F21-15-B3, 5′-TACGGAGATGCTTAGCCGAGC-3′ (282/275/211). The full-length cDNA was isolated using 5′ and 3′ rapid amplification of cDNA ends based on the manufacturer's protocol (SMART RACE cDNA Amplification Kit; Clontech, Palo Alto, CA). The primers were located at 42451 to 42473 and 42783 to 42802 bp in the F19K6 BAC clone, with a 351-bp overlap.

Constructs and Transgenic Arabidopsis thaliana

The ABA2/GIN1 genomic clone (7.7 kb), covering 3 and 2 kb of the 5′ and 3′ flanking regions, respectively, was amplified by PCR, confirmed by sequencing, and cloned into a miniature binary vector (Xiang et al., 1999). The ABA2/GIN1 full-length cDNA was amplified by PCR from a cDNA library (Jang et al., 1997), confirmed by sequencing, and cloned into a binary vector (pSMAB704) under the control of the constitutive 35S promoter to generate 35S::GIN1. The GIN1::β-glucuronidase (GUS) fusion gene was generated by fusing the 3-kb 5′ promoter of GIN1 to GUS at the ATG site. The promoter was obtained by PCR from the genomic clone using the following primers with underlined HindIII and SmaI sites, respectively: 5′-ATCTCACTACGGAAGCTTCTGG-3′ and 5′-GTGACCCCGGGAATA-GCTTTAGCTCCTTAGATCTTC-3′, starting at nucleotides 38638 and 41545, respectively, in the F19K6 BAC clone. The promoter sequence was confirmed by DNA sequencing and cloned into binary vector pSMAB704. The binary vectors were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. The null gin1-3 mutant and wild-type plants were transformed by Agrobacterium using the floral-dip method (Hwang and Sheen, 2001). T1 transgenic plants showing herbicide resistance were confirmed further by PCR and DNA gel blot analyses. More than 100 transgenic plants were obtained for all constructs. Ten independent transgenic lines for each of the cDNA and genomic clones were analyzed further. GUS expression pattern analyses were performed with three randomly chosen transgenic lines in the wild type and gin1-3. T2 and homozygous T3 plants were used to perform complementation tests and GUS expression pattern analyses as described (Jang et al., 2000).

Subcellular Localization of the GIN1–Green Fluorescent Protein Fusion

The GIN1 coding region was amplified by RT-PCR and fused to green fluorescent protein (GFP) in a plant expression vector for protoplast transient expression analyses, as described previously (Hwang and Sheen, 2001). The following primers with a BamHI site at the 5′ end and a StuI site at the 3′ end (underlined) were used: 5′-AAG-AAGATCTAAGGATCCAAAGC-3′ and 5′-ATGCAAGGCCTTCTGAAG-ACTTTAAAGGAGTGG-3′. RT-PCR amplification of mutant coding regions (gin1-2, gin1-3, and aba2-1) was performed to make GFP fusions. The expression of the gin1-3–GFP fusion was not observed in protoplasts, suggesting a null mutation (data not shown).

Protein Sequence Alignment and Phylogenetic Tree Construction

Arabidopsis short-chain dehydrogenase/reductase (SDR)–related sequences (46% identity and 60% similarity) were obtained using the BLAST program from the TAIR World Wide Web site (http://www.arabidopsis.org/Blast), and SDR, 11-β-hydroxysteroid dehydrogenase (HSD), retenoid dehydrogenase, and 15-hydroxyprostaglandin dehydrogenase sequences (30% identity and 50% similarity) from other model systems were obtained using the National Center for Biotechnology Information PSI-BLAST program based on the ABA2/GIN1 sequence (http://www.ncbi.nlm.nih.gov/BLAST). The phylogenic tree was generated using SDR sequences containing motifs I to VI, without the divergent N and C termini, with Clustal X 1.81 (ftp:// ftp-igbmc.u-strasbg.fr/pub/ClustalX) and was displayed with Treeview 1.6.5 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The scale of 0.1 in the unrooted tree (Figure 3) indicates 0.1 amino acid substitutions per site. Arabidopsis SDR2 (62%), SDR3 (62%), SDR4 (61%), and SDR5 (60%) share the highest sequence similarity to SDR1. Based on the Clustal alignment program, Arabidopsis ABA2/GIN1 (AtSDR1) is grouped with tobacco (NtSDR) and cucumber (CsSDR) homologs in the phylogenic tree. AtSDR2 is closely related to maize ZmTS2 and is different from AtSDR3, AtSDR4, and AtSDR5. Arabidopsis 11-β-hydroxysteroid dehydrogenases is closely related to human and mouse 11-β-hydroxysteroid dehydrogenases. The human and mouse 15-hydroxyprostaglandin dehydrogenases and human and mouse retenoid dehydrogenases also are shown in Figure 3.

Expression and Purification of SDR1

For SDR1 expression in Pichia pastoris, ABA2/GIN1 cDNA was cloned into the pPICZC vector to produce a fusion protein with the MYC epitope and polyhistidine tags with an ACC sequence before the initiation ATG codon, according to the manufacturer's instructions (Easy Select Pichia Expression Kit; Invitrogen, Carlsbad, CA). Transformation and expression of recombinant protein were performed as described (Koiwai et al., 2000). The cells were harvested after methanol induction for 48 h. The crude enzyme extract was fractionated with ammonium sulfate (0 to 45% saturation) and dialyzed against 50 mM potassium phosphate buffer, pH 7.5, containing 2.5 mM EDTA and 5 μM leupeptin. Ammonium sulfate–fractionated enzyme samples were purified with the ProBound protein purification kit (Invitrogen) according to the manufacturer's instructions, except that the elution was performed with 50 mM potassium phosphate buffer, pH 7.5, containing 30 mM EDTA. The eluate was dialyzed against 50 mM potassium phosphate buffer, pH 7.5, containing 2.5 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol and applied to a DEAE column (DEAE-5PW; 7.5 mm i.d. × 75 mm; TOSOH, Tokyo, Japan). The adsorbed proteins were eluted with a linear gradient of KCl from 50 mM to 0.5 M in 20 mM potassium phosphate buffer, pH 8.0, containing 2.5 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol. SDR1 activity was recovered in the fraction that eluted at ~0.3 M KCl. The purity of recombinant SDR1 was examined by SDS-PAGE and protein staining (Figure 10C).

SDR1 Enzymatic Assay

The enzyme sample used for the activity assay was the fraction purified on the Ni2+ column. To determine the Km value, reaction mixtures (100 μL) containing 5 μL of enzyme solution (~5 μg of protein), 0.01 to 0.1 mM xanthoxin, 50 mM potassium phosphate buffer, pH 7.5, and 5 mM NAD were incubated at 30°C for 5 to 20 min. After incubation, the reaction was stopped by adding 10 μL of 1 N HCl and 30 μL of 100% methanol, successively. After centrifugation, the supernatant (100 μL) was subjected to HPLC. Activity was assayed by determining the amount of abscisic aldehyde product, as described previously (Koshiba et al., 1996). The coenzyme dependence of the reaction was determined using 5 mM NAD, NADH, NADP, or NADPH. To determine the reaction product, the reaction was performed with 1 mL of reaction mixture using enzyme solutions purified on the DEAE column. The reaction product was partially purified by ether fractionation and HPLC and subjected to gas chromatography–mass spectrometry (GC-MS).

Abscisic Acid Determination

Seedlings (0.5 to 1 g) were homogenized in 80% (v/v) acetone containing 0.1 mg/mL 2,6-di-tert-butyl-4-methylphenol. After adding 13C-abscisic acid (ABA) (Asami et al., 1999) as an internal standard, the homogenate was shaken for 1 h on ice in darkness and then centrifuged (1200g for 10 min) at 4°C. The precipitate was reextracted, and the combined supernatant was evaporated to remove acetone. ABA was partially purified from the residual aqueous solution by partitioning using hexane and ether and then by HPLC. ABA was methylated with diazomethane and analyzed by GC–selected ion monitoring–MS by monitoring 192 (from 13C-ABA) and 190 (from endogenous ABA) fragments.

GC-MS Analysis

GC-MS analysis was performed with a mass spectrometer (GCMS-QP5050; Shimadzu, Kyoto, Japan) coupled to a gas chromatograph (GC-17A; Shimadzu) with a DB-1 capillary column (0.25 mm i.d. × 30 m, film thickness of 0.25 μm; J&W Scientific, Folsom, CA). The injection temperature was set at 250°C, and the inlet pressure was 70 kPa. After injection, the oven temperature was maintained at 80°C for 1 min, increased to 290°C at a rate of 20°C/min, and then kept at 290°C for 5 min. Mass spectra were obtained under the following conditions: electron energy of 1.5 kV, ion source temperature of 250°C, capillary interface temperature of 250°C, and ion range of 50 to 450 (mass-to-charge ratio).

Preparation of Xanthoxic Acid

A cis and trans mixture of 4′-benzoyl xanthoxic acid ethyl ester was synthesized according to the method described previously (Yamamoto and Oritani, 1996). Treatment of this diester with 2.5 equivalent KOH in dry ethanol under reflux conditions gave a potassium salt of the acid. The usual workup gave a cis and trans mixture of xanthoxic acid in high yield. Each isomer was separated by HPLC to give xanthoxic acid.

Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.

Accession Numbers

The accession number for ABA2/GIN1 is At1g52340, and that for the F19K6 BAC clone is AC037424. Accession numbers for other sequences mentioned in this article are as follows: ZmTS2, L20621; HsSDR, NM_016246; MmSDR, NM_008278; DmSDR, AAF51984; CeSDR, Z72508; ScSDR, NC_001143; EcSDR, NC_000913; AtSDR1, AC037424; NtSDR, NTAJ3177; CsSDR, AF286651; AtSDR2 (AL132968, At3g51680); AtSDR3 (AC004411, At2g47130); AtSDR4 (AB026657, At3g29250); AtSDR5 (AB026657, At3g29260); AtHSD, NM_124448; human HSD, NM_005525; mouse HSD, S75207); human HPGD, NM_000860; mouse HPGD, NM_008278; human RDH, NM_003708, XM_006732; and mouse RDH, AY028928, AF013288.

Acknowledgments

We thank P. Rodriguez and M. Bevan for sharing unpublished results, M. Koornneef for advice, J. Ecker for ein2 seeds, and the ABRC (Columbus, OH) for all other ABA and ethylene mutant seeds. H. Ichikawa is thanked for the binary vector pSMAB704, C. Xiang for the miniature binary vector pCB302, and M.R. Willmann for help with the transgenic plants. We also thank Q. Hall for silique pictures, A. Schaeffner for protocols, D. Bongard for BAC DNA sequence annotation, P.-C. Lin for GUS assays, and B. Moore and S.-H. Cheng for help with the confocal microscope. I. Hwang provided a database search. R. Patharkar, G. Tena, and J. Plotnikova are thanked for harvesting and testing transgenic seeds, T. Oritani for kindly providing xanthoxin, and F. Rolland for critical reading of the manuscript. J.S. was supported by grants from the National Science Foundation (IBN-9723610) and the National Institutes of Health (GM60493). T.K. was supported in part by a Grant-in-Aid for Scientific Research (B) (13490024) from the Ministry of Education, Science, Sports, and Culture of Japan. M.S. was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. P.L. was supported by a grant from the Consejo Nacional de Ciencia y Tecnología (31791) and by the Howard Hughes Medical Institute.

Notes

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.006494.

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