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First published online May 1, 2003; 10.1104/pp.103.021725 Plant Physiology 132:830-839 (2003) © 2003 American Society of Plant Biologists Overexpression of AtCPS and AtKS in Arabidopsis Confers Increased ent-Kaurene Production But No Increase in Bioactive Gibberellins1Department of Biology, Box 91000, Duke University, Durham, North Carolina 27708 (C.M.F., C.J.D., T.-p.S.); and Plant Science Center, RIKEN, 2–1 Hirosawa, Wako-shi, Saitama 351–0198, Japan (S.Y., A.H., H.K., Y.K.)
The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.
The plant growth hormone GA is important for processes including seed germination, stem growth, and flower and fruit development. The GA biosynthetic pathway has been characterized, and genes encoding most GA biosynthetic enzymes have been cloned in Arabidopsis and other species (Hedden and Phillips, 2000  ). In the first portion of the pathway, the precursor geranylgeranyl diphosphate (GGDP) is converted to ent-kaurene in a two-step process catalyzed by ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS; Fig. 1). These enzymes are the products of the Arabidopsis genes AtCPS and AtKS, respectively (Sun et al., 1992; Yamaguchi et al., 1998b). Subsequent reactions catalyzed by the cytochrome P450 enzymes ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO) produce ent-kaurenoic acid (KA) and GA12 from ent-kaurene (Helliwell et al., 1999, 2001a). The final portion of the pathway branches from GA12 to form non-13-hydroxylated metabolites and bioactive GA4 or early 13-hydroxylation metabolites and bioactive GA1. These steps are catalyzed by GA 20-oxidase and GA 3-oxidase enzymes (encoded by multiple AtGA20ox and AtGA3ox genes, respectively). GA 13-oxidase, which converts GA12 to GA53 to form the 13-hydroxylated metabolites, has not been identified in any plant species.
Plants that have been treated with an excess of GA hormone have a characteristic GA overdose morphology. This includes increased hypocotyl length, paler leaves, elongated petioles, taller stem height, and earlier flowering time (Jacobsen and Olszewski, 1993
Overexpression experiments have also been attempted with the AtCPS gene of Arabidopsis. Because AtCPS encodes the enzyme catalyzing the first committed step in GA biosynthesis and because it shows a highly regulated, tissue-specific expression pattern (Silverstone et al., 1997a Rate-limiting steps have not been clearly defined for the GA biosynthetic pathway. Therefore, overproduction of enzymes at the beginning of the pathway may be informative because increased flux through the pathway could identify steps at which metabolite accumulates. To determine the effect of increased levels of enzymes catalyzing early steps in GA biosynthesis, we examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both AtCPS and AtKS. We investigated their morphology, ent-kaurene, KA and GA content, response to a GA biosynthesis inhibitor, and expression of downstream GA biosynthetic genes. We report here that CPS and CPS/KS overexpressors (OEs) produce increased levels of ent-kaurene while maintaining normal morphology and WT levels of bioactive GA.
Generation of Overexpression Lines
The reaction catalyzed by CPS is a possible step limiting the flow of metabolite through the GA biosynthetic pathway because the substrate, GGDP, is a common precursor for other products, including carotenoids, the phytol chain of chlorophyll, and the growth hormone abscisic acid (Takahashi et al., 1991
To generate lines overexpressing AtCPS, Ler (WT) plants were transformed with the pGA1-49 CPS overexpression construct (Sun and Kamiya, 1994 To overproduce KS protein in Arabidopsis, a cassette containing the CaMV 35S promoter-TEV-NTR-AtKS cDNA was cloned into the binary vector pDHB321. This vector has the bar gene conferring resistance to BASTA in plants. We introduced this plasmid construct into WT and one CPS OE via Agrobacterium tumefaciens-mediated transformation. In the T2 generation, we selected four and six transgenic lines in WT and CPS OE background, respectively, that segregated approximately 3:1 for BASTA resistance versus sensitivity. Homozygotes were selected by examining segregation at the T3 generation.
Protein levels of all lines were determined by immunoblot analysis, using anti-CPS antisera (Sun and Kamiya, 1994
Although it is not possible to determine the absolute degree of CPS overexpression in our conditions, we used recombinant CPS and KS protein to determine amounts of each enzyme present in the overexpression lines. Dilutions of protein from WT and CPS or KS OEs were compared with known amounts of recombinant protein for quantification. From this method, we estimate that the amount of CPS in line CPS OE-a is approximately 1 ng µg–1 total protein. The amount of KS is estimated to be 0.1 ng µg–1 total protein in WT and 0.5 ng KS µg–1 total protein in line KS OE-a (data not shown).
WT plants treated with exogenous GA and transgenic plants overexpressing the downstream GA biosynthetic gene AtGA20ox1 showed aspects of GA overdose morphology (Huang et al., 1998
The lack of GA overdose morphology in the CPS and/or KS OEs suggests that bioactive GA levels may not be increased in these plants. Plants from selected transgenic lines were grown on soil to rosette stage (d 21 or 22) for combined gas chromatography (GC)-selected ion monitoring (SIM) analysis to determine their levels of kaurenoids and GAs. For initial ent-kaurene measurements, plants were treated with the GA biosynthesis inhibitor paclobutrazol (PAC) 4 d before harvest to promote the accumulation of sufficient levels of ent-kaurene for detection. Because high levels of ent-kaurene were found in the PAC-treated tissue, especially in overexpression lines, measurements were repeated once using untreated rosette tissue to determine normal ent-kaurene accumulation. A total of five lines was used for this analysis: transgenic lines expressing medium or high levels of CPS; CPS OE-b and -a, respectively; KS overexpression line KS OE-a; double overexpression line CPS/KS OE-a; and control WT.
Because ent-kaurene is the immediate product of the reactions catalyzed by CPS and KS, its level of accumulation should suggest whether the overexpressed proteins are functional. The medium (CPS OE-b) and high (CPS OE-a) OEs show a 30- and 1,008-fold increase in ent-kaurene levels, respectively, compared with untreated tissue (Table I). The CPS/KS double overexpression line shows the greatest increase, with 1,888-fold more ent-kaurene than WT. However, ent-kaurene levels in the KS OE line are comparable with WT. This suggests that it is CPS rather than KS that is limiting for ent-kaurene production, which is consistent with reports that AtCPS mRNA is expressed at much lower levels than AtKS mRNA in WT plants (Silverstone et al. 1997; Yamaguchi et al. 1998b
The later portion of the GA biosynthesis pathway branches downstream of GA12. The non-13-hydroxylation branch and the early 13-hydroxylation branch are parallel portions of the pathway that produce bioactive GA4 and GA1, respectively (Fig. 1). We measured GA levels in both branches (Table II). Relative amounts of GA12 in WT and transgenic lines correspond with the trend observed for earlier metabolites. The GA12 level was near WT in KS OE-a, 5- to 9-fold of WT level in the CPS overexpression lines, and 10 times greater than WT in the CPS/KS double OE. Similarly, the non-13-hydroxylated metabolites GA15 and GA24 were somewhat elevated in the CPS lines and in the double OE. Notably, however, levels of GA9, bioactive GA4, and inactivated GA34 and GA51 were similar to WT for all transgenic lines measured.
Overall metabolite levels were higher in the non-13-hydroxylated pathway than in the parallel 13-hydroxylation pathway. This may be expected because GA4 is the primary active GA in Arabidopsis (Talón et al., 1990
PAC impairs GA biosynthesis as a competitive inhibitor of KO (Rademacher, 1991 We compared the effect of PAC on WT and CPS/KS OEs during seedling growth and initiation of flowering. Hypocotyl elongation was assayed with plants grown on plates containing varying concentrations of PAC that still allow germination of all lines tested. Plants were incubated in the dark to promote longer hypocotyl growth for easier measurement. CPS OE-a and CPS/KS OE-a showed high levels of resistance to PAC and maintained long hypocotyls even at 0.5 µM PAC (Fig. 3A). KS OE-a responded similarly to WT, with hypocotyl length decreasing at increasing PAC, whereas CPS OE-b showed an intermediate response between that of WT and high-CPS OEs. CPS overexpression also conferred resistance to PAC during rosette development and transition to flowering. WT plants grown on 1 µM PAC developed a GA-deficient phenotype, with smaller rosettes, dark-green leaves, and delayed flowering (Fig. 3, B and C). KS OEs responded similarly to WT for flowering time and rosette development, whereas CPS OE-b showed only a slight increase in PAC resistance (Fig. 3, B and C). PAC-treated CPS OE-a and CPS/KS OE-a flowered within 1 d of untreated plants and had more resistant rosette development (Fig. 3, B and C). Despite the increased resistance of CPS OE-a and CPS/KS OE-a to PAC, these plants showed delayed inflorescence elongation (Fig. 3C). CPS and CPS/KS OE lines also showed increased PAC resistance at seed germination (data not shown).
Because the greatest difference in metabolite accumulation between the CPS and CPS/KS OEs versus WT occurs between KA and GA12, the reaction catalyzed by KAO may be rate limiting. This could be because endogenous KAO levels are insufficient to handle excess metabolite or because AtKAO mRNA levels are down-regulated in the overexpression lines. Two genes encoding KAO have been identified in Arabidopsis, AtKAO1 and AtKAO2 (Helliwell et al., 2001a
Previous work has shown that the AtGA20ox1 and AtGA3ox1 genes, encoding enzymes that catalyze the final steps in GA biosynthesis, are under negative feedback regulation (Chiang et al., 1995
Although genes encoding the enzymes for most steps in GA biosynthesis have been cloned in Arabidopsis, the regulation of the GA biosynthetic pathway is less well understood. Overproduction of enzymes catalyzing the first committed steps in GA biosynthesis may be one tool to aid our understanding of the control of GA biosynthesis. Because CPS is present at lower levels than downstream biosynthetic enzymes, overexpression of this protein may increase flux through the pathway and show possible rate-limiting steps downstream.
We have generated lines that produce high levels of CPS or KS protein or both CPS and KS in Arabidopsis. CPS and CPS/KS OEs have high levels of the early biosynthetic intermediates ent-kaurene and KA but have WT levels of bioactive GAs and normal morphology. However, KS OEs have WT ent-kaurene levels. This shows that CPS is limiting for ent-kaurene biosynthesis, consistent with data from Arabidopsis and C. maxima, indicating that CPS expression is more limited than KS expression (Silverstone et al., 1997b
The ability of plants to maintain homeostasis of GA hormone levels in the presence of a 1,000-fold increase in ent-kaurene and KA suggests that later steps in GA biosynthesis are tightly regulated. Overexpression of the penultimate enzyme in GA biosynthesis, GA 20-oxidase, does result in increased GA content (Huang et al., 1998 Measurements of the GA biosynthetic intermediates from the overexpression lines give some indication as to which steps in the pathway may be rate limiting. The most significant change in accumulation of metabolite in the OEs as compared with WT occurs from KA to GA12. Lines with high levels of CPS (CPS OE-a and CPS/KS OE-a) accumulate at least 1,000 times more ent-kaurene and KA than WT but only approximately 10 times more GA12 than WT, a 100-fold decrease in accumulation. This difference identifies the portion of the pathway whose reactions are catalyzed by KAO as likely rate-limiting in the CPS and CPS/KS OE plants. RNA levels of AtKAO1 and AtKAO2 are not decreased in the overexpression lines (Fig. 4, A and B), and we cannot rule out the possibility of decreased KAO enzyme amount or activity. However, our data suggest that endogenous KAO levels may be insufficient to process the great increase in KA present in the CPS and CPS/KS OE lines, resulting in high levels of KA but less increase in GA12. Thus, although CPS appears limiting for ent-kaurene biosynthesis, KAO may be limiting for production of middle and later GA intermediates. Plants engineered to overproduce KAO, perhaps in combination with CPS and/or GA20ox, may have a more pronounced increase in bioactive GA than that observed with overexpression of AtGA20ox1 alone. However, it is still unclear whether KAO in WT plants would ever encounter such high substrate levels for its activity to be limiting.
Evidence from plants such as Alpidea amatysia, C. maxima, and Cucurbita pepo suggests that KA can be converted to kaurenolides and also be converted to GA12 (Fukui et al., 1977
Recently, a novel class of GA 2-oxidases has been identified in Arabidopsis (Schomberg et al., 2003
Another possible limitation on metabolite flux through the GA biosynthetic pathway is that of transport of intermediates, subcellularly or across cell types. Although CPS and KS are localized to plastids (Sun and Kamiya, 1994
Work using green fluorescent protein fusion proteins has shown that KO, which converts ent-kaurene to KA, may be localized to the outer face of chloroplasts and the cytoplasm and/or endoplasmic reticulum. The authors suggest that this localization of KO may provide a way for biosynthetic intermediate to move from the plastid to the endoplasmic reticulum (Helliwell et al., 2001b
We found that the overexpression lines contain elevated GA12 and GA53 but normal GA9 and GA20 levels. As discussed for KAO, this could be because limited amounts of GA 20-oxidases cannot handle excess substrates, or because GA 20-oxidation activity is down-regulated in the transgenic lines. We showed that AtGA20ox1 and AtGA3ox1 mRNA levels in the OEs are not decreased (Fig. 4; data not shown), indicating that negative feedback regulation of these genes does not account for the normal active GA levels in the OEs. The lack of negative feedback regulation in the OEs may not be surprising because there is no significant increase in bioactive GAs in these lines. In addition, the unchanged expression of downstream biosynthetic genes indicates that levels of upstream biosynthetic intermediates such as ent-kaurene do not affect transcriptional regulation of AtGA20ox1 or AtGA3ox1. Thus, potential down-regulation of GA 20-oxidation activity would be attributed to changes in protein levels or protein activity of GA 20-oxidases and/or expression of other AtGA20ox genes (Phillips et al., 1995 In conclusion, the ability of plants to maintain GA homeostasis despite the very large increase in early metabolite in the CPS and CPS/KS OEs suggests that many levels of regulation exist to preserve appropriate levels of bioactive GA.
Plant Material and Growth Conditions Experiments were conducted using Arabidopsis of the ecotype Ler. Seeds were stratified at 4°C for 3 to 4 d and sown on soil at 22°C under LDs (16 h of light, 8 h of dark). For plate growth assays, plants were grown on media containing Murashige and Skoog salts plus 2% (w/v) Suc and 0.8% (w/v) agar. Plants for GA analysis and kaurene measurements were grown on soil in LD conditions to d 21 or 22, within 1 to 2 d of the appearance of the first flower bud. Rosettes were then harvested and frozen in liquid nitrogen for storage at –80°C until lyophilization. Additional plants for ent-kaurene analysis were grown on soil to d 21, then drenched with a solution of PAC (Bonzi, Uniroyal, Middlebury, CT) at 8.75 mL Bonzi L–1 (=120 µM active ingredient PAC). Each flat, containing approximately 250 plants, was bottom watered with 1 L of PAC solution. Tissues were harvested at 4 d after treatment and frozen in liquid nitrogen.
CPS overexpression lines were generated using pGA1-49, containing the 2.4-kb AtCPS cDNA under the control of the CaMV 35S promoter and TEV-NTR, in the pBIN19 vector conferring kanamycin resistance (Sun and Kamiya, 1994
For KS OEs, a translational fusion of the AtKS cDNA (2.4 kb) to the CaMV 35S-TEV-NTR was made. We first introduced an AflIII site at the starting ATG codon of the AtKS gene by PCR. A PCR-amplified partial AtKS coding region (0.3 kb), having AflIII and SacI sites at the 5' and 3' ends, respectively, was cloned into the NcoI site (compatible with an AflIII site) and SacI sites of pRTL2 vector (Restrepo et al., 1990 Selection of transformants was based on antibiotic or herbicide resistance. Transgenic plants containing pGA1-49 were screened on Murashige and Skoog with 50 µg mL–1 kanamycin. Plants containing pGA2-9 were screened on soil by spraying with a 1:10,000 (w/v) dilution of gluphosinate ammonium (BASTA; Hoechst Agro-Evo, Berlin) daily for 3 d, beginning after the appearance of the first true leaves (about 1 week). Rescreening at T3 was conducted on plates containing 50 µM gluphosinate ammonium (Crescent Chemicals, Happauge, NY).
For production of KS protein, the pET/AtKS plasmid, containing the His-T7-tagged 2.5-kb AtKS cDNA (Yamaguchi et al., 1998a
Total protein from 3-week-old Arabidopsis rosette tissue was extracted as described by Silverstone et al. (2001
Frozen tissues were ground in a chilled mortar and pestle and transferred to a solution of 80% (v/v) methanol (MeOH). Tissue was filtered and partitioned three times against n-hexane. The n-hexane fraction was evaporated at 12°C to a small volume, applied to a silica gel column, and eluted with n-hexane. Clear fractions were pooled and evaporated for GC-SIM analysis (Auto Mass, JEOL, Tokyo) using a capillary column DB-1 mass spectrometer (J&W Scientific, Folsom, CA). Oven temperature was set to 80°C for the first minute, followed by an increase of 30°C min–1 to 200°C, and then 5°C min–1 to 300°C. ent-Kaurene levels were determined by comparison with peak area of di-deuterated internal standard (257/259).
Lyophilized tissues (0.1–0.5 g dry weight) were extracted with 80% (v/v) aqueous MeOH and homogenized with a blender. ent-[17,17-2H2]kaurenoic acid (KA) was added to the extract as an internal standard. Extracts were evaporated in vacuo, and the acidic ethyl acetate-soluble fraction was obtained after solvent partitioning. Samples were then dissolved in n-hexane and were fractionated by silica gel chromatography. The eluate with n-hexane containing 5% (v/v) acidic ethyl acetate was collected as a fraction containing KA. The KA fraction was evaporated to dryness, dissolved in MeOH, and then purified on a C18 cartridge (Varian, Palo Alto, CA), which was eluted with 90% (v/v) aqueous MeOH. The eluate was evaporated under vacuum and then dissolved in MeOH. The sample was applied to a DEA cartridge (Varian) and eluted with MeOH containing 2% (v/v) acetic acid. The eluate was then purified by HPLC (System Gold, Beckman Instruments, Fullerton, CA) using a C18 column (Capcell Pak C18, 6
Purification and analysis was as described (Gawronska et al., 1995
PAC for plate assays was supplied by Zeneca (Richmond, CA). All PAC solutions contained 0.01% (v/v) Tween 20 to promote penetration of PAC through the seed coat. Control solutions for these experiments also contained 0.01% (v/v) Tween 20. For the hypocotyl growth assay, seeds were surface sterilized and cold treated for 2 d. Ten to 20 seeds per line were sown at d 0 on Murashige and Skoog plates containing each concentration of PAC. After 3 h of light exposure to promote germination, plates were double wrapped in foil, placed in boxes, and incubated at 22°C in darkness. Etiolated hypocotyls were measured on d 6. Seeds for flowering time were sown on 100- x 20-mm petri plates containing either Murashige and Skoog or Murashige and Skoog plus 1 µM PAC. Plates were incubated in growth chambers (22°C in LD), and the position of the plates was rotated daily to account for possible variations in light intensity. Flowering time was determined by the first appearance of flower buds.
RNA extraction from 3-week-old rosettes was as described (Ausebel et al., 1990
For RNA-blot analysis, poly(A+) RNA was purified from 0.5 to 1 mg of total RNA from the same samples used for qPCR. Purification was performed using the PolyATtract mRNA Isolation System (Promega, Madison, WI) according to the manufacturer's protocol. RNA gel-blot analysis was carried out using an antisense RNA probe (Yamaguchi et al., 1998a
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
We thank Andy Phillips (Long Ashton Research Station, University of Bristol, UK) for providing AtGA20ox1 cDNA clone. We are grateful to members of Tai-ping Sun's lab (Duke University, Durham, NC) and to Peter Hedden (IACR Long Ashton Research Station, Department of Agricultural Science, University of Bristol, Long Ashton, UK) for helpful discussions. Received February 4, 2003; returned for revision February 26, 2003; accepted March 5, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.021725.
1 This work was supported by the U.S. Department of Agriculture (grant no. 99–35304–8061) and by the National Science Foundation (grant no. INT–9603418).
2 These authors contributed equally to the paper.
3 Present address: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183–8509, Japan.
4 Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02155. * Corresponding author; e-mail tps{at}duke.edu; fax 919–613–8177.
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ABSTRACT
RESULTS
-hydroxylation of C20-GAs, such as GA12 and GA53, to form inactive GAs, GA110 and GA97, respectively (
x 250 mm; Shiseido, Tokyo). Samples were injected in 20% (v/v) MeOH and were eluted with 20% (v/v) MeOH for 5 min, a 20% to 100% (v/v) gradient of MeOH in water containing 0.1% (v/v) acetic acid for 10 min, and then 100% (v/v) MeOH at a flow rate of 1 mL min–1. The fraction containing KA (21–22 min) was trimethylsilylated with N-methyl-N-trimethylsilyltrifluoroacetamide and then analyzed by GC-SIM. Oven temperature was held at 80°C for the 1st min, then increased to 250°C at 30°C min–1, followed by further increase to 320°C at 5°C minute –1. The content of KA was calculated from peak areas of mass-to-charge ratio 374 (cold KA) and mass-to-charge ratio 376 ([17, 17-2H2]KA). 
