Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana

Abstract

In plants, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of isoprenoids, is compartmentalized: the mevalonate (MVA) pathway, which is localized to the cytosol, is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone; in contrast, the recently discovered MVA-independent pathway, which operates in plastids, is involved in providing the precursors for monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. Specific inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) were used to perturb biosynthetic flux in Arabidopsis thaliana seedlings. The interaction between both pathways was studied at the transcriptional level by using GeneChip (Affymetrix) microarrays and at the metabolite level by assaying chlorophylls, carotenoids, and sterols. Treatment of seedlings with lovastatin resulted in a transient decrease in sterol levels and a transient increase in carotenoid as well as chlorophyll levels. After the initial drop, sterol amounts in lovastatin-treated seedlings recovered to levels above controls. As a response to fosmidomycin treatment, a transient increase in sterol levels was observed, whereas chlorophyll and carotenoid amounts decreased dramatically when compared with controls. At 96 h after fosmidomycin addition, the levels of all metabolites assayed (sterols, chlorophylls, and carotenoids) were substantially lower than in controls. Interestingly, these inhibitor-mediated changes were not reflected in altered gene expression levels of the genes involved in sterol, chlorophyll, and carotenoid metabolism. The lack of correlation between gene expression patterns and the accumulation of isoprenoid metabolites indicates that posttranscriptional processes may play an important role in regulating flux through isoprenoid metabolic pathways.

Fig. 1.

Overview of isoprenoid metabolic pathways localized to the cytosol and to plastids in plants, with an emphasis on the metabolism of chlorophylls, carotenoids, and sterols. The symbol for anabolic reactions is a solid arrow, and catabolic reactions are indicated by arrows with dotted lines. Open arrows depict multiple enzymatic steps, and key metabolites are boxed. Known enzymes involved in isoprenoid metabolism are numbered, and question marks indicate steps for which an enzymatic activity has not yet been demonstrated. Evidence for the branching of the plastidial MVA-independent pathway to yield IPP and dimethylallyl diphosphate independently has recently been reported (27). The following enzymes are represented: 1, 1-deoxy-d-xylulose 5-phosphate synthase; 2, DXR; 3, 2-C-methyl-d-erythritol 4-phosphate cytidyltransferase; 4, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase; 5, 2,4-C-methyl-d-erythritol cyclodiphosphate synthase; 6, 1-hydroxy-2-methyl-2-(E)-butenyl-4-phosphate synthase; 7, 1-hydroxy-2-methyl-2-(E)-butenyl-4-phosphate reductase; 8, acetoacetyl-CoA thiolase; 9, 3-hydroxy-3-methylglutaryl-CoA synthase; 10, HMGR; 11, MVA kinase; 12, phospho-MVA kinase; 13, MVA diphosphate decarboxylase; 14, IPP: dimethylallyl diphosphate isomerase; 15, geranyl diphosphate synthase; 16, farnesyl diphosphate synthase; 17, geranylgeranyl diphosphate synthase; 18, geranylgeranyl reductase; 19, phytoene synthase; 20, phytoene desaturase; 21, ζ-carotene desaturase; 22, lycopene β-cyclase; 23, lycopene ε-cyclase; 24, β-carotene hydroxylase; 25, zeaxanthin epoxidase; 26, violaxanthin de-epoxidase; 27, epoxycarotenoid (neoxanthin) cleavage enzyme; 28, abscisic aldehyde oxidase; 29, glutamyl tRNA synthetase; 30, glutamyl tRNA reductase; 31, glutamate 1-semialdehyde aminotransferase; 32, aminolevulinate dehydratase; 33, porphobilinogen deaminase; 34, uroporphyrinogen synthase; 35, uroporphyrinogen decarboxylase; 36, coproporphyrinogen III oxidase; 37, protoporphyrinogen IX oxidase; 38, Mg-protoporphyrinogen IX chelatase; 39, Mg-protoporphyrinogen IX methyltransferase; 40, protoporphyrinogen cyclase (enzyme activity not yet characterized in plants); 41, protochlorophyllide reductase; 42, chlorophyll synthetase; 43, chlorophyll a oxygenase; 44, chlorophyll b reductase (gene not cloned yet); 45, chlorophyllase; 46, de-chelatase (gene not cloned yet); 47, pheophorbide a oxygenase (gene not yet cloned); 48, red chlorophyll catabolite reductase; 49, squalene synthase; 50, squalene monooxygenase; 51, cycloartenol synthase; 52, cycloartenol C24 methyltransferase; 53, 24-methylenecycloartenol C-4 methyl oxidase; 54, cycloeucalenol cycloisomerase; 55, sterol C14 reductase; 56, obtusifoliol 14-demethylase; 57, C-8,7 sterol isomerase; 58, sterol C-methyltransferase 2; 59, sterol C-4 methyl oxidase; 60, sterol C5-desaturase; 61, sterol Δ7 reductase; 62, sterol C24-reductase; and 63, sterol C22-desaturase.

Fig. 2.

Effects of inhibitor treatments on A. thaliana seedlings; phenotypic characteristics of control seedlings for lovastatin experiments (A); lovastatin-treated seedlings (B); control seedlings for fosmidomycin experiments (C); fosmidomycin-treated seedlings (D); levels of sterols (•), chlorophylls (▾), and carotenoids (▪) (± standard deviation) in the presence of lovastatin, and fosmidomycin (E and F). Values were calculated on 1 μg/g fresh-weight basis and were normalized (100% for mock control). Fresh weight per seedling did not change significantly with the various treatments.

Fig. 3.

Effects of lovastatin (A) and fosmidomycin (B) treatments on the expression of the genes encoding HMGR (HMG1 gene) and DXR (± standard deviation) in A. thaliana seedlings as indicated by RT-PCR analysis.