The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis

Abstract

Plant isoprenoids are synthesized via two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastid nonmevalonate pathway. The Escherichia coli IspH (LytB) protein is involved in the last step of the nonmevalonate pathway. We have isolated an Arabidopsis (Arabidopsis thaliana) ispH null mutant that has an albino phenotype and have generated Arabidopsis transgenic lines showing various albino patterns caused by IspH transgene-induced gene silencing. The initiation of albino phenotypes rendered by IspH gene silencing can arise independently from multiple sites of the same plant. After a spontaneous initiation, the albino phenotype is systemically spread toward younger tissues along the source-to-sink flow relative to the initiation site. The development of chloroplasts is severely impaired in the IspH-deficient albino tissues. Instead of thylakoids, mutant chloroplasts are filled with vesicles. Immunoblot analysis reveals that Arabidopsis IspH is a chloroplast stromal protein. Expression of Arabidopsis IspH complements the lethal phenotype of an E. coli ispH mutant. In 2-week-old Arabidopsis seedlings, the expression of 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), IspD, IspE, IspF, and IspG genes is induced by light, whereas the expression of the IspH gene is constitutive. The addition of 3% sucrose in the media slightly increased levels of DXS, DXR, IspD, IspE, and IspF mRNA in the dark. In a 16-h-light/8-h-dark photoperiod, the accumulation of the IspH transcript oscillates with the highest levels detected in the early light period (2-6 h) and the late dark period (4-6 h). The expression patterns of DXS and IspG are similar to that of IspH, indicating that these genes are coordinately regulated in Arabidopsis when grown in a 16-h-light/8-h-dark photoperiod.

Figure 1.

MVA and nonMVA pathways in plants. HMG-CoA, 3-Hydroxy-3-methylglutaryl CoA; MVA, mevalonic acid; MVAP, mevalonic acid 5-phosphate; MVAPP, mevalonic acid 5-diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; Mt, mitochondrion; UQ, ubiquinone; GA-3-P, glyceraldehyde 3-phosphate; DOXP, 1-deoxy-d-xylulose-5-phosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-d-erythritol; CDP-ME2P, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate; ME-2,4cPP, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; GGPP, geranylgeranyl diphosphate; GA, gibberellic acid; PQ, plastoquinone; ABA, abscisic acid. Enzymes of the MVA pathway: HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, MVA kinase; PMK, MVAP kinase; MDD, MVAPP decarboxylase. Enzymes of the nonMVA pathway: DXS, DOXP synthase; DXR, DOXP reductoisomerase; CMS, CDP-ME synthase; CMK, CDP-ME kinase; MCS, ME-2,4cPP synthase; HDS, HMBPP synthase; HDR, HMBPP reductase. The names of their corresponding genes are indicated on the left.

Figure 2.

Phenotypic analysis of Arabidopsis ispH-1 mutants. A, Segregation of ispH-1 homozygous (albino) plants. B, A 6-week-old ispH-1 plant grown on Murashige and Skoog plus Suc medium in a plantcon. C and D, Transmission electron micrographs of wild-type (C) and ispH-1 mutant (D) chloroplasts. Sections are from the first leaves of 2-week-old Arabidopsis plants grown in tissue culture. Scale bars, 1 cm (A and B); 500 nm (C and D).

Figure 3.

A, Schematic diagram of the Arabidopsis IspH gene. Arrows indicate EcoRV restriction sites. Black boxes indicate exons. The T-DNA (white triangle) is not drawn to scale. B, Northern and immunoblot analyses. Total RNA (10 μg) and proteins (20 μg) extracted from 2-week-old wild-type (WT) and ispH-1 plants were used for northern (top) and immunoblot (bottom) analyses to detect the IspH mRNA and IspH protein, respectively. After detection of the IspH mRNA, the membrane was stripped and reprobed with 18S rDNA as a control (middle). C, Expression pattern of the Arabidopsis IspH gene. Total RNA (10 μg) extracted from 6-week-old wild-type Arabidopsis plants grown in soil was used for northern-blot analysis. R, Roots; L, leaves; St, stems; F, flowers; Si, siliques. The ethidium bromide-stained agarose gel of the same samples is shown at the bottom. D, Eight-day-old Arabidopsis wild-type (WT), ispH-1, and 35S:IspH cDNA complemented (Com) seedlings. E, Genomic Southern analysis (EcoRV digested). The arrow indicates the ispH-1 mutant allele and the arrowhead indicates the 35S:IspH transgenic allele in a complemented (Com) line.

Figure 4.

Arabidopsis 35S:IspH cDNA transgene-induced gene silencing. A, Schematic diagram of a 35S:IspH cDNA construct. B, Representative primary transformants of 35S:IspH Arabidopsis after BASTA treatment. Red arrows indicate IspH-silencing plants. C, Northern-blot analysis of IspH mRNA. D, Immunoblot analysis of IspH protein. WT, Wild-type rosette leaves; G, green tissues of IspH-silenced leaves; W, white tissues of IspH-silenced leaves.

Figure 5.

Systemic spread of IspH gene silencing in Arabidopsis. A, Initiation and subsequent systemic spread of IspH gene silencing occurred in rosette leaves, stems (primary and lateral), and siliques independently. B, Pale green phenotype of an IspH-silenced plant. Progeny derived from this plant segregate green, pale green, and various albino patterns randomly. C, Albino inflorescence of an IspH-silenced plant. D, Green and partially green cauline leaves attached to an IspH-silenced (albino) stem. The arrow indicates the albino tip of an IspH-silenced silique. E, Wild-type (left) and various albino phenotypes of IspH-silenced siliques. F, Four representative T3 homozygous lines. Lines 1, 3, and 4 randomly segregate silenced (with various albino patterns) and nonsilenced (green) plants. All plants in line 2 are green. G to I, Systemic spread of the albino phenotype toward developing rosette leaves. The plant shown in G, 3 d (H) and 6 d (I) later. J, Initiation of IspH gene silencing in the base (indicated by an arrow) of an expanding rosette leaf. The plant shown in J, 3 d (K) and 9 d (L) later.

Figure 6.

Transmission electron micrographs. Ultrastructure of chloroplasts from (A) wild-type rosette leaves, (B) green, and (C) albino tissues of an IspH-silencing leaf. D to I, Ultrastructure of chloroplasts from a pale green to pale yellow boundary between the green and albino tissue of an IspH-silencing leaf. Scale bars, 500 nm. A 5-week-old Arabidopsis IspH gene-silencing plant used for transmission electron microscopy is shown on the top.

Figure 7.

Arabidopsis IspH protein is localized in the chloroplast stroma. Fifteen micrograms of total proteins (T), chloroplast stromal proteins (Chl.S), and chloroplast membrane proteins (Chl.M) were used for immunoblot analysis. After detection with the IspH antibody, the membrane was stripped and reprobed with OE33 antibody to detect the membrane-localized protein. The same protein samples were analyzed in a replicate membrane to detect the stroma-localized rbcS and the membrane-localized LHC-II chlorophyll a/b-binding protein.

Figure 8.

Arabidopsis IspH complements the E. coli ispH mutant. The E. coli ispH mutant strain MG1655 ara<>IspH was able to grow on Luria-Bertani media containing 0.2% Ara, but not on media containing 0.2% Glc (left). After transformation with the Arabidopsis IspH cDNA (pQE-AtIspH) and, as a control, with the empty vector (pQE) alone, the resulting strains were tested for growth on media containing 0.2% Glc (right).

Figure 9.

Northern-blot analysis of Arabidopsis nonMVA pathway genes. Total RNA (10 μg) extracted from 2-week-old Arabidopsis seedlings treated with light or dark for 48 h in the presence or absence of 3% Suc was used to detect the expression of nonMVA pathway genes. Steady-state levels of DXS, DXR, IspD, IspE, IspF, and IspG mRNAs are significantly increased by light. Suc, Sucrose; Man, mannitol. The ethidium bromide-stained agarose gel of the same samples is shown at the bottom.

Figure 10.

Expression patterns of Arabidopsis nonMVA pathway genes in a normal day/night cycle. A, Arabidopsis seedlings were grown on tissue culture plates (Murashige and Skoog plus 3% Suc) in a 16-h-light/8-h-dark cycle, and samples were collected every 2 h on days 13 and 14. Total RNA (10 μg) extracted from these samples was used to detect the expression patterns of the nonMVA pathway genes. The light/dark expression patterns of Arabidopsis ASN1 and rbcS were also detected in the same RNA samples as controls. The ethidium bromide-stained agarose gel of the same samples is shown at the bottom. B, Quantification of northern blots in A showing distinct expression patterns of the Arabidopsis nonMVA pathway genes. The signals were quantified using the National Institutes of Health Image 1.62 software and normalized to the loading control 25S rRNA. After normalization, the highest mRNA level of each gene was set at 1.0. The line charts generated by Microsoft Graph software represent the relative mRNA levels of the nonMVA pathway genes, ASN1, and rbcS in A.