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1-Deoxy-d-xylulose-5-phosphate Synthase, a Limiting Enzyme for Plastidic Isoprenoid Biosynthesis in Plants

2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês

10.1074/jbc.m100854200

1083-351X

Juan M. Estévez, Araceli Cantero, Andreas Reindl, Stuart Reichler, Patricia León,

Lipid metabolism and biosynthesis

The initial step of the plastidic 2C-methyl-d-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-d-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production. The initial step of the plastidic 2C-methyl-d-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-d-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production. abscisic acid 1-deoxy-d-xylulose 5-phosphate 1-deoxy-d-xylulose-5-phosphate synthase isopentenyl diphosphate dimethylallyl diphosphate acetate/mevalonate 2C-methyl-d-erythritol 4-phosphate 1-deoxy-d-xylulose-5-phosphate reductoisomerase gibberellins polymerase chain reaction reverse-transcriptase-PCR 4-morpholineethanesulfonic acid Isoprenoids are a group of biologically active molecules that number in the tens of thousands. Members of this diverse group of natural products are found in all organisms. In higher plants, isoprenoids participate in a wide variety of biological functions such as photosynthesis, respiration, growth, cell cycle control, plant defense, and adaptation to environmental conditions. Specific examples include photosynthetic pigments (chlorophylls and carotenoids), hormones (abscisic acid (ABA),1 gibberellins (GA), cytokinins, and brassinosteroids), a side chain of the electron transporter (plastiquinone), structural components of membranes (phytosterols), and antimicrobial agents (phytoalexins). Beyond these plant-specific functions, many plant isoprenoids have been shown to have industrial and medical importance. The plant-produced isoprenoids β-carotene (provitamin A) and α-tocopherol (vitamin E) are both basic nutrients required for the maintenance of human health (1Hirschberg J. Curr. Opin. Biotechnol. 1999; 10: 186-191Crossref PubMed Scopus (87) Google Scholar, 2Shintani D. DellaPenna D. Science. 1998; 282: 2098-2100Crossref PubMed Scopus (436) Google Scholar, 3Römer S. Fraser P.D. Kiano J.W. Shipton C.A. Misawa N. Schuch W. Bramley P.M. Nat. Biotechnol. 2000; 18: 666-669Crossref PubMed Scopus (350) Google Scholar, 4Ye X. Al-Babili S. Klöti A. Zhang J. Lucca P. Beyer P. Potrykus I. Science. 2000; 287: 303-305Crossref PubMed Scopus (1667) Google Scholar). Another plant-produced isoprenoid, Taxol, is used as a chemotherapeutic agent in the treatment of cancer (5Blagosklonny M.V. Fojo T. Int. J. Cancer. 1999; 83: 151-156Crossref PubMed Scopus (333) Google Scholar). Moreover, drugs that block isoprenoid production in Plasmodium falciparum are being evaluated as anti-malarial agents (6Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Türbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1021) Google Scholar). Industrial uses of isoprenoids include products such as colorants, fragrances, and flavorings (7Lange B.M. Croteau R. Curr. Opin. Plant Biol. 1999; 2: 139-144Crossref PubMed Scopus (93) Google Scholar). A detailed understanding of isoprenoid biosynthetic pathways and their regulation is essential to fully exploit these and future uses of isoprenoids.Isoprenoids are derived by consecutive condensations of five-carbon precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). From these common precursors, the biosynthetic pathways of the various isoprenoids diverge. The work of several groups has demonstrated that in plants two distinct pathways synthesize IPP (Fig. 1). The acetate/mevalonate (MVA) pathway (8Newman J.D. Chappell J. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 95-106Crossref PubMed Scopus (205) Google Scholar, 9Bach T.J. Boronat A. Campos N. Ferrer A. Vollack K.-U. Crit. Rev. Biochem. Mol. 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Naturforsch. 1997; 52: 15-23Crossref Scopus (133) Google Scholar, 18Eisenreich W. Schwarz M. Cartayrade A. Arigoni D. Zenk M. Bacher A. Chem. Biol. 1998; 5: 221-233Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Although there is evidence that some limited exchange occurs in plants between the cytoplasmic and plastidic pools of IPP, each pathway appears to produce unique isoprenoids (15Lichtenthaler H.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 47-65Crossref PubMed Scopus (1080) Google Scholar, 19Lichtenthaler H.K. Rohmer M. Schwender J. Physiol. Plant. 1997; 101: 643-652Crossref Google Scholar).Whereas all of the genes involved in the MVA pathway have been identified, only the first genes of the plant MEP pathway have been published (Fig. 1). The first step in the MEP pathway involves a transketolase-type condensation reaction of pyruvate and glyceraldehyde 3-phosphate to yield 1-deoxy-d-xylulose-5-phosphate (DXP). This reaction is catalyzed by DXP synthase (DXS). Genes encoding DXS have been cloned and characterized in Escherichia coli (20Sprenger G.A. Schörken U. Wiegert T. Grolle S. De Graaf A.A. Taylor S.V. Begley T.P. Bringer-Meyer S. Sahm H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12857-12862Crossref PubMed Scopus (411) Google Scholar,21Lois L.M. Campos N. Putra S.R. Danielsen K. Rohmer M. Boronat A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2105-2110Crossref PubMed Scopus (345) Google Scholar), Mentha × piperita (22Lange B.M. Wildung M.R. McCaskill D. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2100-2104Crossref PubMed Scopus (341) Google Scholar), Capsicum annuum (23Bouvier F. d'Harlingue A. Suire C. Backhaus R.A. Camara B. Plant Physiol. 1998; 117: 1423-1431Crossref PubMed Scopus (162) Google Scholar), Synechococcus leopoliensis (24Miller B. Heuser T. Zimmer W. FEBS Lett. 1999; 460: 485-490Crossref PubMed Scopus (36) Google Scholar),Lycopersicon esculentum (25Lois L.M. Rodrı́guez-Concepción M. Gallego F. Campos N. Boronat A. Plant J. 2000; 22: 503-513Crossref PubMed Google Scholar), Streptomyces (26Kuzuyama T. Takagi M. Takahashi S. Seto H. J. Bacteriol. 2000; 182: 891-897Crossref PubMed Scopus (118) Google Scholar), and Arabidopsis thaliana (27Mandel M.A. Feldmann K.A. Herrera-Estrella L. Rocha-Sosa M. León P. Plant J. 1996; 9: 649-658Crossref PubMed Scopus (251) Google Scholar, 28Estévez J.M. Cantero A. Romero C. Kawaide H. Jiménez L.F. Kuzuyama T. Seto H. Kamiya Y. León P. Plant Physiol. 2000; 124: 95-103Crossref PubMed Scopus (205) Google Scholar). In plants, the DXP produced by this reaction is utilized in plastidic IPP biosynthesis as well as in the production of thiamin and pyridoxol (29Julliard J.H. C. R. Acad. Sci. ( Paris ). 1992; 314: 285-290Google Scholar, 30Julliard J.H. Douce R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2042-2045Crossref PubMed Scopus (112) Google Scholar). The subsequent steps of the MEP pathway have been shown to be specific for IPP production, and the genes coding for the next four steps have been identified in both bacteria and plants (Fig. 1). The first step specific for IPP production is the transformation of DXP to MEP by the enzyme DXP reductoisomerase (DXR) (6Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Türbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1021) Google Scholar, 31Takahashi S. Kuzuyama T. Watanabe H. Seto H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9879-9884Crossref PubMed Scopus (441) Google Scholar, 32Lange B.M. Croteau R. Arch. Biochem. Biophys. 1999; 365: 170-174Crossref PubMed Scopus (149) Google Scholar, 33Schwender J. Müller C. Zeidler J. Lichtenthaler H.K. FEBS Lett. 1999; 455: 140-144Crossref PubMed Scopus (137) Google Scholar, 34Miller B. Heuser T. Zimmer W. FEBS Lett. 2000; 481: 221-226Crossref PubMed Scopus (51) Google Scholar). MEP is subsequently converted into 2C-methyl-d- erythritol 2,4-cyclodiphosphate by the consecutive activities of three independent enzymes as shown in Fig. 1 (35Rohdich F. Wungsintaweekul J. Fellermeier M. Sagner S. Herz S. Kis K. Eisenreich W. Bacher A. Zenk M.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11758-11763Crossref PubMed Scopus (240) Google Scholar, 36Rohdich F. Wungsintaweekul J. Eisenreich W. Richter G. Schuhr C.A. Hecht S. Zenk M.H. Bacher A. Prot. Natl. Acad. Sci. U. S. A. 2000; 97: 6451-6456Crossref PubMed Scopus (98) Google Scholar, 37Lüttgen H. Rohdich F. Herz S. Wungsintaweekul J. Hecht S. Schuhr C.A. Fellermeier M. Sagner S. Zenk M.H. Bacher A. Eisenreich W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1062-1067Crossref PubMed Scopus (209) Google Scholar, 38Rohdich F. Wungsintaweekul J. Lüttgen H. Fischer M. Eisenreich W. Schuhr C.A. Fellermeier M. Schramek N. Zenk M.H. Bacher A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8251-8256Crossref PubMed Scopus (105) Google Scholar, 39Herz S. Wungsintaweekul J. Schuhr C.A. Hecht S. Luttgen H. Sagner S. Fellermeier M. Eisenreich W. Zenk M.H. Bacher A. Rohdich F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2486-2490Crossref PubMed Scopus (240) Google Scholar). The final steps leading to IPP remain unknown.Identifying the different enzymes involved in the synthesis of IPP is only part of the work necessary to characterize such a complex biosynthetic pathway. Another fundamental aspect is the identification of the limiting steps in the pathway. Large changes in the level of a non-limiting enzyme can be made with little or no effect on the quantity of the final product; therefore, one of the most fruitful strategies for altering isoprenoid content will be those that focus on the rate-limiting enzymes (40Kinney A.J. Curr. Opin. Plant Biol. 1998; 1: 173-178Crossref PubMed Scopus (35) Google Scholar). In the case of the MEP pathway, recent studies in bacteria (24Miller B. Heuser T. Zimmer W. FEBS Lett. 1999; 460: 485-490Crossref PubMed Scopus (36) Google Scholar, 26Kuzuyama T. Takagi M. Takahashi S. Seto H. J. Bacteriol. 2000; 182: 891-897Crossref PubMed Scopus (118) Google Scholar, 34Miller B. Heuser T. Zimmer W. FEBS Lett. 2000; 481: 221-226Crossref PubMed Scopus (51) Google Scholar) and tomato (25Lois L.M. Rodrı́guez-Concepción M. Gallego F. Campos N. Boronat A. Plant J. 2000; 22: 503-513Crossref PubMed Google Scholar) suggest that DXS is a rate-limiting enzyme.In this article we report on the effects of altering DXS levels, the first gene in plastidic isoprenoid synthesis in plants. Our previous work demonstrated that, in A. thaliana, DXS is encoded by the CLA1 gene (28Estévez J.M. Cantero A. Romero C. Kawaide H. Jiménez L.F. Kuzuyama T. Seto H. Kamiya Y. León P. Plant Physiol. 2000; 124: 95-103Crossref PubMed Scopus (205) Google Scholar). In order to explore the participation of DXS in plastidic isoprenoid synthesis in plants, the enzyme levels were increased or decreased in Arabidopsis plants. Analysis of several transgenic lines showed that plants overexpressing DXS had increased levels of isoprenoids such as chlorophylls, tocopherols, carotenoids, ABA, and GA. Moreover, plants with suppressed levels of DXS had decreased amounts of all of these products. The fact that alterations in DXS levels lead to changes of various isoprenoid end products demonstrates that DXS is one of the limiting steps in the production of plastidic IPP and, therefore, of isoprenoids in higher plants. Each group of plastidic isoprenoids has a distinct biosynthetic pathway that could be manipulated; however, our findings indicate that the output of these pathways could be restricted by the amount of IPP that is produced in the plastids.DISCUSSIONBecause isoprenoids are such ubiquitous and essential compounds, there have been intensive efforts to understand the pathways that lead to their production. All isoprenoids are derived from the precursor molecules IPP and DMAPP, which are produced in plants by either the cytoplasmic MVA or plastidic MEP pathways (19Lichtenthaler H.K. Rohmer M. Schwender J. Physiol. Plant. 1997; 101: 643-652Crossref Google Scholar, 59Lange B.M. Rujan T. Martin W. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13172-13177Crossref PubMed Scopus (637) Google Scholar). There has been substantial progress in the identification of the biosynthetic steps of the MEP pathway in the past few years. However, knowing the reaction steps involved in the MEP pathway is only the first step to fully understanding it. Fundamental aspects that need to be addressed are the regulatory and control points of the pathway. If DXS is a limiting enzyme of the MEP pathway, then altering its level will affect the quantity of IPP. Analogously, if the amount of plastidic IPP is limiting in the production of isoprenoids, alterations in the IPP level will have an effect on overall isoprenoid levels. One way to define experimentally the rate-limiting steps of a biosynthetic pathway is by using reverse genetics to make changes in specific sites of the pathway and then monitoring the corresponding changes in the end products (60Furbank R.T. Taylor W.C. Plant Cell. 1995; 7: 797-807Crossref PubMed Google Scholar). The experiments described in this article were designed to determine whether the first enzyme in the MEP pathway, DXS, is a limiting part of the production of plastidic IPP. Additionally, if DXS is limiting, we wanted to evaluate the effects of altering its level on seed germination, plant growth, and isoprenoid abundance.The general finding for three divergent isoprenoid pathways is that by raising the level of DXS, the levels of isoprenoids are raised, and when the level of DXS is lowered, so are the levels of isoprenoids. Because the changes in DXS levels lead to changes in isoprenoid abundance, we propose that DXS is one of the limiting enzymes in the MEP pathway. It is likely that other co-limiting enzymes for each specific isoprenoid are present in the plant MEP pathway or further downstream, as we did not observe a linear relationship between changes in DXS and its end products. Additional limiting enzymes would also explain the differences observed in the relative increases among the isoprenoids monitored in this work. This is the case for chlorophyll and carotenoid content where increases in their levels are more restricted than others. To identify other limiting enzymes of the MEP pathway will require further analyses when the complete enzymatic steps are known and the relative effects of changing the levels of other MEP pathway enzymes have been evaluated. Recently, the MEP pathway has been intensively studied in bacteria where it was found that DXS also is as a limiting enzyme, and DXR is not (26Kuzuyama T. Takagi M. Takahashi S. Seto H. J. Bacteriol. 2000; 182: 891-897Crossref PubMed Scopus (118) Google Scholar, 34Miller B. Heuser T. Zimmer W. FEBS Lett. 2000; 481: 221-226Crossref PubMed Scopus (51) Google Scholar, 52Harker M. Bramley P.M. FEBS Lett. 1999; 448: 115-119Crossref PubMed Scopus (132) Google Scholar). This conclusion is also supported by recent work on tomato fruit ripening. Lois et al. (25Lois L.M. Rodrı́guez-Concepción M. Gallego F. Campos N. Boronat A. Plant J. 2000; 22: 503-513Crossref PubMed Google Scholar) found that DXS was limiting for carotenoid production over the previously identified limiting step of carotenoid biosynthesis (PSY1). They reasoned that DXS was limiting the amount of IPP available for carotenoid synthesis because even when there were increases inPSY1 transcript levels, there were no increases in carotenoids without increases in DXS. The accumulated evidence indicates that DXS is one of the limiting steps in the MEP pathway of plants as well as in bacteria.In this work we analyzed isoprenoids that are synthesized at very low levels such as hormones, and isoprenoids that are required in large quantities such as chlorophylls and carotenoids. In both cases a moderate change in the DXS level produced differences in the levels of the final isoprenoid products. Despite the fact that the biosynthetic pathway of GA has been intensively studied, there is no direct evidence that these hormones are actually synthesized via the MEP pathway. Our data suggest that GA biosynthesis depends, at least in part, on the IPP that comes from the MEP pathway. However, based on the expression of the GA4 gene, the cla1-1 mutant seems to contain active GA, which suggests that an additional source of IPP exists. Whether this IPP is the result of the import of cytosolic IPP (15Lichtenthaler H.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 47-65Crossref PubMed Scopus (1080) Google Scholar) or if it comes from other sources remains to be established. Other groups have also looked at increasing or decreasing the levels of the hormones ABA (58Thompson A.J. Jackson A.C. Symonds R.C. Mulholland B.J. Dadswell A.R. Blake P.S. Burbidge A. Taylor I.B. Plant J. 2000; 23: 363-374Crossref PubMed Google Scholar, 61Frey A. Audran C. Marin E. Sotta B. Marion-Poll A. Plant Mol. Biol. 1999; 39: 1267-1274Crossref PubMed Scopus (111) Google Scholar) and GA (62Eriksson M.E. Israelsson M. Olsson O. Moritz T. Nat. Biotechnol. 2000; 18: 784-788Crossref PubMed Scopus (393) Google Scholar, 63Huang S.S. Raman A.S. Ream J.E. Fujiwara H. Cerny R.E. Brown S.M. Plant Physiol. 1998; 118: 773-781Crossref PubMed Scopus (179) Google Scholar, 64Coles J.P. Phillips A.L. Croker S.J. Garcı́a-Lepe R. Lewis M.J. Hedden P. Plant J. 1999; 17: 547-556Crossref PubMed Scopus (245) Google Scholar) by modulating the levels of enzymes in their individual post-IPP biosynthetic pathways. These studies have obtained relatively large changes in hormone levels with phenotypes complementing the proposed functions of these hormones, which demonstrate additional key regulatory steps in these biosynthetic pathways.Much research has been devoted to increasing the levels of the isoprenoids α-carotene, β-carotene (3Römer S. Fraser P.D. Kiano J.W. Shipton C.A. Misawa N. Schuch W. Bramley P.M. Nat. Biotechnol. 2000; 18: 666-669Crossref PubMed Scopus (350) Google Scholar, 65Shewmaker C.K. Sheehy J.A. Daley M. Colburn S. Ke D.Y. Plant J. 1999; 20: 401-412Crossref PubMed Google Scholar, 66Fray R.G. Wallace A. Fraser P.D. Valero D. Hedden P. Bramley P.M. Grierson D. Plant J. 1995; 8: 693-701Crossref Scopus (296) Google Scholar), and α-tocopherol (2Shintani D. DellaPenna D. Science. 1998; 282: 2098-2100Crossref PubMed Scopus (436) Google Scholar) due, in part, to the importance of these molecules as precursors to vitamins needed by mammals. When total carotenoid content was increased (65Shewmaker C.K. Sheehy J.A. Daley M. Colburn S. Ke D.Y. Plant J. 1999; 20: 401-412Crossref PubMed Google Scholar, 66Fray R.G. Wallace A. Fraser P.D. Valero D. Hedden P. Bramley P.M. Grierson D. Plant J. 1995; 8: 693-701Crossref Scopus (296) Google Scholar), there were concomitant decreases in other isoprenoids. The results of these studies also support that the amount of IPP is limiting for isoprenoid production. In contrast, when only the relative quantity of isoprenoid end products was changed such as α-carotene to β-carotene (3Römer S. Fraser P.D. Kiano J.W. Shipton C.A. Misawa N. Schuch W. Bramley P.M. Nat. Biotechnol. 2000; 18: 666-669Crossref PubMed Scopus (350) Google Scholar) or γ-tocopherol to α-tocopherol (2Shintani D. DellaPenna D. Science. 1998; 282: 2098-2100Crossref PubMed Scopus (436) Google Scholar), there were no other effects noted. By changing the amount of DXS and thus IPP levels, we are reporting the first instance in plants where a general increase or decrease in multiple plastidic isoprenoids was observed. Because moderate alterations in DXS levels (38–172x0025;) lead to changes in all of the isoprenoids tested, it is apparent that the IPP from the MEP pathway is also limiting for the production of plastidic isoprenoids. The limiting role of IPP availability in isoprenoid production implies that to increase an individual isoprenoid without decreasing other isoprenoids requires a concurrent increase in IPP production.Even though the levels of the different isoprenoids increase or decrease according to the level of DXS, the various isoprenoids do not change equally showing the complexity of the isoprenoid biosynthetic pathways that diverge from plastidic IPP. These results, together with the studies mentioned above wherein amounts of individual isoprenoids could be modulated through manipulation of genes in their post-IPP biosynthetic pathways, demonstrate that each of these post-IPP biosynthetic pathways has its own set of limiting and regulatory steps.Aside from the measured changes in isoprenoid content, the DXS-overexpressing and -suppressed transgenic plants had close to normal phenotypes when grown in germination medium or in soil under optimal conditions. Significant differences were observed in the growth rates of the 8- and 26-day-old transgenic plants but not in the final plant size. These differences are not likely to be due solely to the observed changes in GA levels because changes of this hormone result in either dwarf or giant phenotypes (67Ross J.J. Murfet I.C. Reid J.B. Physiol. Plant. 1997; 100: 550-560Crossref Google Scholar). A possible explanation for the growth rate effects in the transgenic lines is that the plastidic IPP pathway receives its substrates directly from the Calvin cycle; therefore, changing the amount of IPP produced in these plants might have a direct effect on photosynthetic carbon availability to other pathways in the cell. Therefore, we hypothesized that the differences in growth rates are likely due to a pleiotropic effect of changes in carbon metabolism and hormone levels. On the other hand, the differences in germination rates can likely be directly correlated to the effects of either raised or lowered ABA levels. ABA plays a major role in setting and maintaining dormancy in seeds (56Koornneef M. Karssen C.M. Meyerowitz E.M. Somerville C.R. Seed Dormancy and Germination. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 313-334Google Scholar). The DXS-suppressed plants with less ABA content germinated rapidly, whereas the DXS-overexpressing plants had poor germination rates. These results are very similar to those obtained for ABA mutants of the post-IPP biosynthetic pathway (58Thompson A.J. Jackson A.C. Symonds R.C. Mulholland B.J. Dadswell A.R. Blake P.S. Burbidge A. Taylor I.B. Plant J. 2000; 23: 363-374Crossref PubMed Google Scholar, 61Frey A. Audran C. Marin E. Sotta B. Marion-Poll A. Plant Mol. Biol. 1999; 39: 1267-1274Crossref PubMed Scopus (111) Google Scholar). In addition to the germination rates, transgenic plants containing 4-fold more ABA do not display any other phenotypes when grown under normal conditions. In contrast to what was reported in tomato, we could not observe overguttation or chlorosis in the leaves of these plants, when grown under normal conditions (58Thompson A.J. Jackson A.C. Symonds R.C. Mulholland B.J. Dadswell A.R. Blake P.S. Burbidge A. Taylor I.B. Plant J. 2000; 23: 363-374Crossref PubMed Google Scholar). It is possible that the levels of ABA in the transgenic lines are not sufficient to generate these phenotypes in Arabidopsis or that a specific growth condition could be required to display these phenotypes. We believe that a more detailed analysis of these plants could provide additional information about ABA regulation and function in plants. For example, the proposed regulation between biosynthesis and catabolism of ABA seems insufficient to prevent the ABA increments observed in the overexpressing plants. These results are similar to what was found with the ectopic expression of the tomato 9-cis-epoxycarotenoid dioxygenase enzyme, involved in the ABA biosynthetic pathway (58Thompson A.J. Jackson A.C. Symonds R.C. Mulholland B.J. Dadswell A.R. Blake P.S. Burbidge A. Taylor I.B. Plant J. 2000; 23: 363-374Crossref PubMed Google Scholar). Altering DXS levels, and hence IPP, manifested itself phenotypically in changes in growth rates, but not the final plant size, as well as having an effect on germination rates.Varying the quantity of DXS may also lead to differences in the amounts of thiamin and pyridoxol because the product of DXS, DXP, is a precursor to these molecules as well as IPP. However, it is unlikely that either thiamin or pyridoxol deficiency gives rise to the observed phenotypes of these transgenic plants because the measurements of isoprenoid content and seedling size were performed in the presence of media containing vitamin supplements. As suggested in the initial studies of the cla1-1 mutant (27Mandel M.A. Feldmann K.A. Herrera-Estrella L. Rocha-Sosa M. León P. Plant J. 1996; 9: 649-658Crossref PubMed Scopus (251) Google Scholar, 28Estévez J.M. Cantero A. Romero C. Kawaide H. Jiménez L.F. Kuzuyama T. Seto H. Kamiya Y. León P. Plant Physiol. 2000; 124: 95-103Crossref PubMed Scopus (205) Google Scholar), alterations in the quantity of vitamins cannot account for the phenotypes that were observed in the transgenic plants.Finally, since many isoprenoids are useful in medical, nutritional, or industrial applications and our ability to manipulate successfully metabolic pathways in plants continues to improve, isoprenoids are becoming a prime target for the production of commercially viable transgenic plants. By having isolated and characterized DXS (27Mandel M.A. Feldmann K.A. Herrera-Estrella L. Rocha-Sosa M. León P. Plant J. 1996; 9: 649-658Crossref PubMed Scopus (251) Google Scholar, 28Estévez J.M. Cantero A. Romero C. Kawaide H. Jiménez L.F. Kuzuyama T. Seto H. Kamiya Y. León P. Plant Physiol. 2000; 124: 95-103Crossref PubMed Scopus (205) Google Scholar), the first gene in the MEP pathway of plants, we wanted to know if DXS was one of the limiting enzymes of the MEP pathway as it is in bacteria (24Miller B. Heuser T. Zimmer W. FEBS Lett. 1999; 460: 485-490Crossref PubMed Scopus (36) Google Scholar, 26Kuzuyama T. Takagi M. Takahashi S. Seto H. J. Bacteriol. 2000; 182: 891-897Crossref PubMed Scopus (118) Google Scholar, 34Miller B. Heuser T. Zimmer W. FEBS Lett. 2000; 481: 221-226Crossref PubMed Scopus (51) Google Scholar). We tested this idea by manipulating DXS levels in transgenic plants. We observed changes in the levels of a wide variety of isoprenoids, and these increases or decreases in isoprenoid levels followed the levels of DXS. From these results we conclude that DXS catalyzes one of the limiting steps of the MEP pathway. Also, since changes in DXS levels exert their effect on isopren