Research ArticleBIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

The Arabidopsis IspH Homolog Is Involved in the Plastid Nonmevalonate Pathway of Isoprenoid Biosynthesis

Ming-Hsiun Hsieh, Howard M. Goodman

Published June 2005. DOI: https://doi.org/10.1104/pp.104.058735

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.

Isoprenoids are the largest group of natural products found in living organisms. Among the important isoprenoids are compounds such as steroid hormones in mammals, carotenoids and chlorophylls in plants, and ubiquinone or menaquinone in bacteria. Still others are medically important for human health, e.g. vitamins, hormones, and anticancer agents such as Taxol (Sacchettini and Poulter, 1997).

All isoprenoids are derived from a basic five-carbon unit, isopentenyl diphosphate (IPP), and its allyl isomer dimethylallyl diphosphate (DMAPP). For decades, the mevalonate (MVA) pathway was believed to be the only route to synthesize IPP and DMAPP. However, recent studies have uncovered an alternative nonmevalonate (nonMVA) pathway for isoprenoid biosynthesis (Rohmer et al., 1993; Eisenreich et al., 1998, 2001; Lichtenthaler, 1999; Rohdich et al., 2001; Rodriguez-Concepcion and Boronat, 2002; Rohmer, 2003). Most, if not all, enzymes involved in the nonMVA pathway have been identified in Escherichia coli (Sprenger et al., 1997; Lois et al., 1998; Takahashi et al., 1998; Rohdich et al., 1999, 2002, 2003; Herz et al., 2000; Luttgen et al., 2000; Hecht et al., 2001; Adam et al., 2002). In the first step, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) converts pyruvate and glyceraldehyde-3-phosphate to 1-deoxy-d-xylulose 5-phosphate (DXP), which also serves as a biosynthetic precursor of vitamins B1 (thiamine) and B6 (pyridoxal; White, 1978; Sprenger et al., 1997). DXP is converted to 2C-methyl-d-erythritol 4-phosphate (MEP) by the 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR or IspC). MEP is then converted to IPP and DMAPP in consecutive steps catalyzed by 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (CMS or IspD), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (CMK or IspE), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS or IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS or IspG), and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR or IspH; Fig. 1). Because MEP is the first committed precursor in the pathway, the nonMVA pathway is also known as the MEP pathway.

Figure 1.
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.

The nonMVA pathway has been found in a broad range of organisms, including bacteria, green algae, and higher plants (Eisenreich et al., 1998, 2001; Lichtenthaler, 1999; Cunningham et al., 2000; Rohdich et al., 2001; Rodriguez-Concepcion and Boronat, 2002). In plants, the MVA and nonMVA pathways are compartmentalized in the cytoplasm and plastid, respectively. Sesquiterpenes, sterols, and polyterpenes are derived from the cytosolic MVA pathway, whereas isoprene, phytol, carotenoids, and plant hormones GA and abscisic acid are synthesized via the plastid nonMVA pathway (Fig. 1). The Arabidopsis genome contains genes encoding homologs of E. coli nonMVA pathway enzymes and the deduced amino acid sequences all possess a transit peptide for chloroplast localization, consistent with their predicted role in the biosynthesis of plastid isoprenoids (Rodriguez-Concepcion and Boronat, 2002).

Previous studies have shown that Arabidopsis plants (cla1-1 mutants) with a null mutation in the DXS gene are albino (Mandel et al., 1996; Estevez et al., 2000, 2001). Analyses of the flanking genomic DNA sequences of a collection of Arabidopsis T-DNA or transposon-tagged seedling lethal lines have identified albino mutants in the DXS, IspC (DXR), and IspD genes, although these lines have not been further characterized (Budziszewski et al., 2001). Levels of photosynthetic pigments are dramatically reduced in the Arabidopsis IspD antisense lines (Okada et al., 2002). The albino phenotype was also observed in Nicotiana benthamiana leaves using tobacco rattle virus (TRV)-IspG- and TRV-IspH-induced gene silencing (Page et al., 2004). Recent studies on Arabidopsis chloroplast biogenesis (clb) albino mutants revealed that the clb4 and clb6 mutants are caused by the loss of function of the IspG and IspH genes, respectively (Gutierrez-Nava et al., 2004; Guevara-Garcia et al., 2005). These observations suggest that plants carrying loss-of-function mutations in any of the nonMVA pathway genes may have a visible pigmentation phenotype.

To isolate plant nonMVA pathway mutants, we generated Arabidopsis T-DNA insertion lines and screened for plants showing pale green or albino phenotypes. One of the isolated Arabidopsis albino mutants is caused by a T-DNA insertion in a gene that encodes a protein with significant similarity to E. coli IspH (or LytB). Consistent with the albino phenotype observed in the null mutant, Arabidopsis IspH gene-silencing plants show pale green to various albino patterns. Levels of IspH mRNA are dramatically reduced in the IspH-silenced albino tissues. We also provide experimental evidence that the Arabidopsis IspH protein is localized in the chloroplast stroma. A complementation test with an E. coli ispH mutant further confirms that the Arabidopsis IspH protein functions as a nonMVA pathway enzyme involved in the biosynthesis of plastid isoprenoids.

The biosynthesis of plastid isoprenoids is directly linked to photosynthesis. We have thus examined the effects of light and Suc on the expression of nonMVA pathway genes in Arabidopsis. In addition, it has been suggested that the biosynthesis and emission of volatile plant isoprenoids are derived from the plastid nonMVA pathway (Lichtenthaler, 1999; Sharkey and Yeh, 2001; Zeidler and Lichtenthaler, 2001). The emission of some volatile plant isoprenoids and the expression of some terpene synthase genes are regulated diurnally or nocturnally (Loreto et al., 1996; Kolosova et al., 2001; Lu et al., 2002; Dudareva et al., 2003; Martin et al., 2003). We also studied the expression patterns of Arabidopsis nonMVA pathway genes under a 16-h-light/8-h-dark photoperiod. Several distinct diurnal expression patterns were observed among the Arabidopsis nonMVA pathway genes. The accumulation of DXS, IspG, and IspH transcripts oscillates in a similar pattern during the 16-h-light/8-h-dark cycle.

RESULTS

Phenotypic Analysis of the Arabidopsis ispH-1 Mutant

We isolated the albino ispH-1 mutant by screening a collection of Arabidopsis T-DNA insertion lines. Genetic analysis and thermal asymmetric interlaced-PCR revealed that the albino line 3a234 contains two copies of T-DNA in two different loci, IspH and At3g46440, which were further segregated as two different lines. Homozygous ispH-1 plants are albino and progeny from a self-pollinated heterozygous plant segregate green and albino plants in a 3:1 ratio on a nonselective medium, i.e. the albino phenotype is inherited as a recessive mutation (Fig. 2A). The ispH-1 mutant seedlings exhibit a purple-tinted phenotype superimposed on the albino phenotype when grown on the medium containing Suc (Fig. 2A). The purple coloration begins to fade about 1 week after germination on this medium. The ispH-1 albino plant can develop a normal root system, rosette leaves, an inflorescence with cauline leaves, and flower-like structures that never mature into normal flowers when grown on tissue culture medium (Fig. 2B).

Figure 2.
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).

To study the effect of the ispH-1 mutation on chloroplast development, leaf sections of Arabidopsis wild-type and ispH-1 plants were examined by transmission electron microscopy. In contrast to the lens-shaped wild-type chloroplast (Fig. 2C), the ispH-1 mutant chloroplasts are usually round, oval, or irregularly shaped (Fig. 2D; data not shown). In addition, the mutant chloroplasts completely lack thylakoids and contain large vesicles (Fig. 2D). In ispH-1 mutants, total chlorophylls and carotenoids are less than 1% and 2%, respectively, of their amounts in the wild type (Table I).

View this table:
Table I.

Photosynthetic pigment content of wild-type Arabidopsis and ispH-1 mutants

Values shown are μg/g fresh weight ± se.

Molecular Characterization and Complementation of the ispH-1 Locus

Analysis of the flanking genomic DNA sequences revealed that the Arabidopsis ispH-1 mutant has a T-DNA insertion in the seventh exon of the IspH gene (Fig. 3A). Northern and immunoblot analyses showed that the IspH mRNA and protein were undetectable in the ispH-1 mutant (Fig. 3B). These results suggest that ispH-1 is a null mutant. In 6-week-old wild-type Arabidopsis plants, the IspH transcript was detected in all tissues analyzed (Fig. 3C). To prove that the defective ispH-1 locus is responsible for the albino phenotype, we restored the wild-type phenotype by introducing into the mutant a full-length IspH cDNA transcribed from a cauliflower mosaic virus 35S promoter. The phenotype of a representative complementation line is shown in Figure 3D. Genomic DNA-blot analysis was used to verify that the complemented plants contained a (homozygous) ispH-1 mutant allele and a 35S:IspH transgene (Fig. 3E). These results confirm that the albino phenotype is caused by disruption of the IspH gene.

Figure 3.
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.

Arabidopsis 35S:IspH cDNA Transgene-Induced Gene Silencing

Attempts to create Arabidopsis IspH overexpression lines resulted in some primary transformants showing pale green or various albino phenotypes (Fig. 4, A and B). In plants, some transgenes may cause a coordinated silencing of the transgene and homologous host genes (Mlotshwa et al., 2002). It is possible that the IspH gene is silenced in the albino tissue. To test this, total RNA and protein extracted from green and albino tissues of the transgenic plants were examined by northern and immunoblot analysis. The steady-state levels of IspH mRNA and protein are higher in the transgenic green tissue than in the wild type, whereas the IspH mRNA and protein are not detectable in the albino tissue (Fig. 4, C and D). These results indicate that IspH is overexpressed in the green tissue and is silenced in the albino tissue of the same plant.

Figure 4.
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.

Initiation and Systemic Spread of IspH Gene Silencing

The visual albino phenotype that is a result of IspH gene silencing serves as a marker for observing the initiation and systemic spread of transgene-induced gene silencing in Arabidopsis. The initiation of IspH gene silencing is spontaneous and stochastic; it may arise at various developmental stages and several independent initiations may even occur in the same plant. For instance, the albino phenotype may appear independently in rosette leaves, stems, and siliques (Fig. 5A). After the initiation step, somehow the IspH gene-silencing signal(s) is systemically spread toward developing tissues so that younger tissues that develop above the initiation site will be affected (Fig. 5, A–C). Expanding cauline leaves, at the time of initiation, either are not or are only partially affected, leading to a phenotype where a green leaf and a partially green leaf are attached to an albino stem (Fig. 5D). Sometimes the apical region of a silenced inflorescence may remain green, which indicates that the IspH gene is not always silenced in the meristematic regions (Fig. 5, A, D, and L). In siliques, IspH gene silencing can be localized in the base, in the tip, in the middle, or at both ends independently and gradually spreads throughout the entire silique (Fig. 5, A, D, and E). The random initiation and systemic spread of the albino phenotype in siliques indicate that cells in developing siliques may not have a distinct source-to-sink status for silencing signals as in leaves or stems. In T3 homozygous lines, the mixed progeny of nonsilenced (green) and silenced plants with various albino patterns segregate randomly (Fig. 5F).

Figure 5.
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.

When the initiation of IspH gene silencing is localized in rosette leaves during the vegetative stage, leaves that have expanded before the initiation will not be affected (Fig. 5, G–I). If the initiation occurs in rosette leaves during the transition from vegetative to reproductive stage, the green inflorescence tip has a chance to develop flowers and siliques before the entire inflorescence becomes albino (Fig. 5, J–L). Neither initiation nor systemic spread of IspH gene silencing was observed in fully expanded rosette leaves.

Dynamics of Thylakoids in the IspH-Silencing Tissues

In a partially silenced rosette leaf, chloroplasts in the green cells accumulate more starch granules than the comparable wild type (Fig. 6, A and B), whereas in the IspH-silenced albino tissue, chloroplasts are highly vesiculated (Fig. 6C). Because the basal part of an expanding leaf is composed of younger tissues, it is possible that these vesicles are derived from undifferentiated chloroplasts. During the systemic spread of IspH gene silencing in a leaf, a narrow boundary line of pale green to pale yellow forms between the green and the albino tissue. Transmission electron microscopy reveals that various types of chloroplasts exist in this region (Fig. 6, D–I). Chloroplasts of pale green tissues close to the nonsilenced green part of the leaf have highly differentiated thylakoids, but most of the stroma lamellae are discontinuous and stacked thylakoids are thicker than the wild type (Fig. 6D). By contrast, chloroplasts of pale yellow tissues close to the albino part of the leaf have only a few differentiated thylakoids (Fig. 6, E and F), mixed vesicles and loosening thylakoids (Fig. 6G), small vesicles (Fig. 6H), or large vesicles (Fig. 6I). The IspH-silencing chloroplasts also contain densely stained globule (lipid-droplet) aggregates (Fig. 6, D, E, G, and I). Since the systemic spread of the albino phenotype starts from the initiation site toward developing tissues, these chloroplasts may represent a broad range of undifferentiated, partially differentiated, and fully differentiated chloroplasts that are affected by photooxidation caused by various levels of IspH gene silencing. The vesicular structures and densely stained globule aggregates observed in these chloroplasts may be derived from the precursor components or breakdown products of thylakoids.

Figure 6.
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.

Arabidopsis IspH Is a Chloroplast Stromal Protein

Alignment of IspH amino acid sequences from plants (Arabidopsis and Adonis), cyanobacterium, and E. coli indicates that both plant IspH proteins have an extra N-terminal sequence with the features of a chloroplast transit peptide (data not shown; Botella-Pavia et al., 2004). Chloroplast fractionation and immunoblot analysis showed that the Arabidopsis IspH protein is associated with the soluble fraction (Fig. 7), which is indicative of localization in the stroma. Control assays were performed to detect the membrane-localized OE33 and light-harvesting complex (LHC)-II proteins and the soluble, stroma-localized small subunit of Rubisco (rbcS) in the same samples (Fig. 7).

Figure 7.
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.

Arabidopsis IspH Complements the E. coli ispH Mutant

To test whether the Arabidopsis IspH protein has similar enzymatic activity to its E. coli counterpart, we performed a complementation assay with an E. coli ispH mutant. In E. coli ispH mutant strain MG1655 ara<>ispH, the endogenous IspH gene was replaced by a kanamycin-resistant cassette and a single copy of IspH was present on the chromosome under the control of the PBAD promoter (McAteer et al., 2001). Because the IspH gene is essential for survival, the E. coli ispH mutant cannot grow in the absence of arabinose (Ara; Fig. 8, left). Growth of the E. coli MG1655 ara<>ispH mutant on the medium containing Glc was restored successfully by transformation of the pQE-AtIspH plasmid but not by the empty pQE-30 vector (Fig. 8, right).

Figure 8.
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).

Light Induction of the nonMVA Pathway Genes

It has been shown that expression of Arabidopsis DXS and DXR (IspC) genes is induced by light (Mandel et al., 1996; Carretero-Paulet et al., 2002). We thus examined the effects of light/dark and Suc on the expression of Arabidopsis IspH and other nonMVA pathway genes. Two-week-old Arabidopsis seedlings grown under normal light conditions (16-h-light/8-h-dark cycle) were subsequently placed in continuous light or dark for 48 h. During the 48-h dark or light treatments, plants were grown in media containing 0% Suc, 3% Suc, or 3% mannitol. The nonmetabolizable sugar mannitol was included as an osmotic control. Total RNA extracted from these samples was used for northern-blot analysis to detect the steady-state mRNA levels of the nonMVA pathway genes. Compared to the dark treatments, light significantly increased the levels of DXS, DXR, IspD, IspE, IspF, and IspG mRNAs (Fig. 9). In the dark, the addition of 3% Suc in the media slightly increased the accumulation of DXS, DXR, IspD, IspE, and IspF transcripts (Fig. 9; compare 0% and 3% Suc in the dark). This Suc induction is not due to an osmotic change because the treatment with mannitol has no significant effect on levels of DXS, DXR, IspD, IspE, and IspF mRNAs (Fig. 9; compare 0% and 3% mannitol in the dark). In contrast to the rest of the nonMVA pathway genes, the steady-state levels of IspH mRNA are relatively constant in response to treatments with light/dark or Suc (Fig. 9).

Figure 9.
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.

Expression Patterns of the nonMVA Pathway Genes in a Normal Day/Night Cycle

To further investigate whether expression of the nonMVA pathway genes is coordinately regulated, we compared the day/night expression patterns of these genes in 13- and 14-d-old Arabidopsis seedlings (Fig. 10). Interestingly, several distinct diurnal expression patterns were observed in the Arabidopsis nonMVA pathway genes. The expression patterns of DXS, IspG, and IspH are similar during the 16-h-light/8-h-dark cycle. Peak levels of DXS, IspG, and IspH mRNA were detected in the early period of the light cycle (2–6 h in the light) and in the late period of the dark cycle (6–8 h in the dark). An additional peak appeared at the end of the light cycle (16 h), which is more evident in relative mRNA levels of DXS and IspG and less obvious in that of IspH (Fig. 10B). Oscillations in DXR, IspD, IspE, and IspF mRNA accumulation also occurred during the light/dark cycle. In contrast to DXS, IspG, and IspH, peak levels of IspD mRNA appeared in the late period of the light cycle (12–16 h). The highest levels of DXR, IspE, and IspF mRNA were also detected in the late period (14 h) of the light cycle. An additional peak in the early period (2–6 h) of the light cycle is evident in the relative mRNA levels of IspE and less obvious in those of DXR and IspF. Interestingly, the expression of all nonMVA pathway genes is significantly repressed during the transition from light to dark (Fig. 10; compare light 16 h to dark 2 h in each cycle). The same RNA samples were used to detect the light/dark expression patterns of Arabidopsis ASN1 (encoding Asn synthetase) and rbcS (encoding Rubisco small subunit) as controls. The expression of Arabidopsis ASN1 is induced by dark and repressed by light (Lam et al., 1998). During a normal 16-h-light/8-h-dark cycle, peak levels of ASN1 mRNA appear in the middle of the light cycle and in the dark cycle (Hsieh, 1998). The expression of Arabidopsis rbcS is induced by light (Dedonder et al., 1993) and oscillates during a light/dark cycle (Pilgrim and McClung, 1993).

Figure 10.
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.

DISCUSSION

The E. coli LytB protein was originally identified as one of the components involved in penicillin tolerance and control of the stringent response (Gustafson et al., 1993). Studies on a lytB mutant of the cyanobacterium Synechocystis strain PCC 6803 provided evidence of a role for LytB in the nonMVA pathway of isoprenoid biosynthesis (Cunningham et al., 2000). Mutagenesis studies in E. coli also suggested that the LytB protein is involved in the nonMVA pathway (Altincicek et al., 2001; McAteer et al., 2001). Recent in vivo and in vitro experiments have confirmed that E. coli LytB catalyzes the last reaction of the nonMVA pathway, a branching step that separately produces IPP and DMAPP in a ratio of 5:1 to 6:1 (Adam et al., 2002; Altincicek et al., 2002; Rohdich et al., 2002, 2003). E. coli LytB was thus renamed IspH (Adam et al., 2002).

Here, we report the identification and characterization of an Arabidopsis albino mutant that has a null mutation in the IspH gene. The phenotype of the ispH-1 mutant and the IspH gene-silencing lines and the localization of the IspH protein are in accord with a role for Arabidopsis IspH in plastid isoprenoid biosynthesis. Consistent with our studies, Page et al. (2004) have shown that leaves of N. benthamiana with the TRV-silenced IspH gene also have an albino phenotype. The clb mutant clb6-1 carries a mutation in the Arabidopsis IspH gene (Gutierrez-Nava et al., 2004; Guevara-Garcia et al., 2005). The plastid nonMVA pathway is the primary contributor for the biosynthesis of chlorophylls and carotenoids, which are essential components for chloroplast development and plant growth. In IspH-deficient albino cells, the biosynthesis of photosynthetic pigments is impaired and, thus, thylakoids cannot fully develop, which, in turn, results in an accumulation of large vesicles inside the mutant chloroplast.

The phenomenon of transgene-induced gene silencing was first uncovered as coordinate silencing (cosuppression) of both the transgene and the homologous plant gene in petunia (Napoli et al., 1990; Van der Krol et al., 1990). It was later shown to occur at the posttranscriptional level (De Carvalho et al., 1992; Van Blokland et al., 1994). Posttranscriptional gene silencing (PTGS) in plants is mechanistically similar to RNA interference in animals (Fire et al., 1998). Despite being avidly studied in recent years, the epigenetic mechanisms of the initiation, propagation, and maintenance of transgene-induced gene silencing are still largely unknown. It has been suggested that the initiation of PTGS could correspond to highly transcribed single-transgene copies or depend on the transgene producing a particular form of RNA above a threshold level (Vaucheret et al., 2001). Because the expression of the transgene or the accumulation of the particular form of RNA varies in each cell, the initiation of PTGS in transgenic plants is stochastic and localized. Our observations on the initiation of Arabidopsis IspH gene silencing further support the notion that it may occur spontaneously. For instance, multiple initiation sites may arise independently in the same transgenic plant (Fig. 5A). Regardless of the same genotype in the IspH transgenic T3 homozygous lines, silenced (albino) and nonsilenced (green) progeny still segregate randomly (Fig. 5F). Moreover, the locations of initiation sites in the IspH-silenced lines vary from plant to plant and are unpredictable. These variations in triggering IspH gene silencing may reflect the different expression levels of the IspH transgene or the variable abundance of a particular form of IspH mRNA in each cell.

The systemic spread of the IspH-silenced albino phenotype toward younger developing tissues, including the apical meristem (Fig. 5, A, C, and G–L), suggests that the transmission of IspH-silencing signals is unidirectional and of high efficiency. In addition, there is no obvious temporal difference in the appearance of albino phenotypes between vein and nonvein tissues during the spread of IspH gene silencing (Fig. 5, G–L). The resulting IspH-silenced tissues are uniformly photobleached rather than variegated. The direction of systemic spread of IspH gene silencing parallels the flow of metabolic source to sink in leaves and stems. In siliques, however, the spread of silencing signals is stochastic (Fig. 5E). The processes involved in carpel and fruit development are not well understood in Arabidopsis (Ferrándiz et al., 1999). It is likely that the metabolic status (e.g. source versus sink) between the tip and the base of siliques is not as distinct as those in leaves. In plants, several lines of evidence indicate that the cell-to-cell systemic spread of PTGS occurs through plasmodesmata and phloem (Palauqui et al., 1997; Jorgensen et al., 1998; Voinnet et al., 1998; Fagard and Vaucheret, 2000; Lucas et al., 2001; Vance and Vaucheret, 2001; Hamilton et al., 2002; Klahre et al., 2002; Mlotshwa et al., 2002; Himber et al., 2003; Mallory et al., 2003; Yoo et al., 2004), and 21- to 25-nucleotide small interfering RNAs have been considered as likely candidates for the systemic silencing signal (Hamilton and Baulcombe, 1999; Himber et al., 2003; Yoo et al., 2004). The recently identified pumpkin (Cucurbita maxima) phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1) has been proposed to be involved in small RNA trafficking (Yoo et al., 2004). A transmembrane protein SID-1 (systemic RNA interference deficient) required for systemic RNA silencing has been identified in Caenorhabditis elegans (Winston et al., 2002; Feinberg and Hunter, 2003). However, there are no apparent homologs of CmPSRP1 and SID-1 in the Arabidopsis genome. Further studies on IspH gene-silencing lines may help uncover the molecular components involved in the initiation and systemic spread of gene silencing in Arabidopsis.

Taking advantage of the systemic spread of the albino phenotype from the initiation site toward developing tissues, we have observed a series of morphological changes in the chloroplasts of IspH gene-silencing cells (Fig. 6, D–I). The integration of the LHCs, which are mainly composed of chlorophylls, carotenoids, and apoproteins, is important for the development of thylakoids and the formation of grana stacking (Bartley and Scolnik, 1995; Von Wettstein et al., 1995; Simidjiev et al., 2000). Thus, the development of the chloroplast in the IspH-silencing cells may be arrested at various stages depending on the levels of silencing. Because thylakoids are mainly composed of galactolipids containing highly unsaturated fatty acids, chloroplast membranes are very sensitive to photooxidative damage (Dormann et al., 1999; Vothknecht and Westhoff, 2001). Carotenoids can protect the photosynthetic apparatus from photooxidative damage (Bartley and Scolnik, 1995; Niyogi, 1999). With the gradual loss of carotenoids in the IspH gene-silencing tissues, thylakoids may lose this protection under light and suffer various levels of photooxidative damage. Since carotenoids have essential functions in photosynthesis and photoprotection, those chloroplasts observed in IspH-silencing tissues may not represent the different developmental stages of wild-type chloroplasts.

Although Arabidopsis IspH only shares about 24% identity (approximately 40% similarity) with the E. coli protein at the amino acid level, expression of Arabidopsis IspH complements the E. coli ispH mutant (Fig. 8). The E. coli IspH protein is a reductase that possesses a dioxygen-sensitive [4Fe-4S] cluster (Wolff et al., 2003). Amino acid sequence alignment reveals that the Cys residues that may be involved in iron-sulfur cluster formation and the His residues that may be involved in proton-transfer reactions (Adam et al., 2002) are also conserved in the Arabidopsis IspH protein (Botella-Pavia et al., 2004). These results indicate that the Arabidopsis and E. coli IspH proteins may share similar enzymatic mechanisms in the biosynthesis of IPP and DMAPP.

In addition to functional analysis and subcellular localization of the Arabidopsis IspH protein, we also characterized the expression and regulation of the Arabidopsis IspH gene. The Arabidopsis IspH transcripts are detected in all parts of adult plants, indicating that the IspH protein has an essential function throughout the entire plant (Fig. 3C). The expression of the Arabidopsis IspH gene in both photosynthetic and nonphotosynthetic tissues supports the notion that the nonMVA pathway is involved in synthesizing a variety of isoprenoids in plants. Consistent with their roles in synthesizing carotenoids and chlorophylls for photosynthesis, the Arabidopsis nonMVA pathway genes are highly expressed in light and most are low in the dark (Fig. 9). The only exception is the IspH gene, whose expression is constitutive regardless of the continuous light/dark treatments in 2-week-old Arabidopsis plants. Interestingly, it has been shown that the expression of the IspH gene is up-regulated during Arabidopsis seedling deetiolation and levels of Arabidopsis IspH mRNA are significantly higher in 3-d-old seedlings grown in continuous light than those of dark-grown seedlings (Botella-Pavia et al., 2004). In addition, Guevara-Garcia et al. (2005) have shown that the transcript levels of the Arabidopsis nonMVA pathway genes are modulated during development with their lowest levels detected in 3-d-old seedlings. Coordinated regulation of all Arabidopsis nonMVA pathway genes only occurs during early developmental stages (between 3 and 6 d) and the accumulation kinetics differ for each gene later in development (Guevara-Garcia et al., 2005). These results, together with our studies, indicate that the expression of the Arabidopsis IspH gene may be differentially regulated by light during different developmental stages.

Since the initial substrates of the nonMVA pathway, pyruvate and glyceraldehyde 3-phosphate, may directly derive from glycolysis and photosynthesis in the chloroplast, carbon metabolites may affect the expression of the nonMVA pathway genes. We have found that the expression of the DXS, DXR, IspD, IspE, and IspF genes is slightly induced by Suc in the dark, whereas the presence of Suc in the light has no additive effects beyond the light induction of the nonMVA pathway genes. These results suggest that at least some of the Arabidopsis nonMVA pathway genes are subjected to metabolic regulation.

Although the expression of the IspH gene is not affected by prolonged (48-h) light or dark treatment (Fig. 9), levels of IspH mRNA oscillate during a 16-h-light/8-h-dark cycle in 2-week-old Arabidopsis plants (Fig. 10). Among the nonMVA pathway genes, DXS and IspG share the most similar expression patterns with IspH during a normal light/dark cycle. Interestingly, expression of the Arabidopsis DXS and IspH genes is coordinately regulated during deetiolation (Botella-Pavia et al., 2004). The first enzyme of the nonMVA pathway, DXS, has been proposed to be a limiting enzyme for the biosynthesis of plastid isoprenoids in Arabidopsis (Estevez et al., 2001). Studies on overexpression of tomato IspH cDNA in Arabidopsis plants led to the conclusion that plant IspH protein also plays a key role in controlling the biosynthesis of plastid isoprenoids (Botella-Pavia et al., 2004). Our studies show that expression of DXS and IspH is also coordinately regulated during a normal light/dark cycle in 2-week-old Arabidopsis plants. Consistent with these results, the diurnal expression of Arabidopsis DXS and IspH genes has also been detected by using microarray analysis (Harmer et al., 2000; Schaffer et al., 2001). Our observations of several distinct diurnal expression patterns of nonMVA pathway genes support the notion that some specific regulatory mechanisms may exist among these genes in 2-week-old Arabidopsis. It will be interesting to further test whether the expression of Arabidopsis nonMVA pathway genes is regulated by circadian rhythm. Recent studies by Guevara-Garcia et al. (2005) have shown that the nonMVA pathway is regulated posttranscriptionally. Thus, it is important to investigate whether levels of the nonMVA proteins oscillate in similar patterns as their corresponding transcripts during a normal day/night cycle.

The MVA pathway in animal cells is regulated at multiple levels, including transcriptional, posttranscriptional, and a complex feedback regulatory system (Goldstein and Brown, 1990). Because the nonMVA pathway genes have been uncovered just recently, little is known about their regulation in plants. It will be very useful if we could develop techniques to directly measure the activities of the nonMVA pathway enzymes in plants. Further studies on how the Arabidopsis nonMVA pathway enzymes are regulated and how the pathway is integrated into the upstream and downstream pathways will provide insights into the complex regulatory network of isoprenoid biosynthesis in plants.

The existence of two independent IPP biosynthetic pathways inside a plant cell raises an interesting question as to whether and, if so, how these two pathways interact with each other. It has been suggested that interactions between the cytosolic MVA pathway and the plastid nonMVA pathway may exist in plants (Eisenreich et al., 1998, 2001; Lichtenthaler, 1999; Kasahara et al., 2002; Nagata et al., 2002; Rodriguez-Concepcion and Boronat, 2002; Bick and Lange, 2003; Laule et al., 2003; Rohmer, 2003). The cross-flow of the compartmentalized isoprenoids may depend on the plant species and has been estimated to be less than 1% in intact plants under physiological conditions (Eisenreich et al., 2001). Recent studies by Laule et al. (2003) suggest that cross-talk between Arabidopsis MVA and nonMVA pathways may occur mainly as posttranscriptional processes. Bick and Lange (2003) have shown that the unidirectional transport of IPP and geranyl diphosphate from plastids to cytosol occurs in plants. The albino lethal phenotype of the ispH-1 mutant implies that the influx of cytosolic isoprenoids into chloroplasts does not occur or, at best, is very limited in Arabidopsis. The reported albino phenotype of Arabidopsis mutants with disrupted DXS (i.e. cla1-1), DXR (IspC), IspD, and IspG (i.e. clb4) genes and the inhibitor fosmidomycin-treated plants also support this notion (Mandel et al., 1996; Budziszewski et al., 2001; Rodriguez-Concepcion and Boronat, 2002; Gutierrez-Nava et al., 2004). In addition, Arabidopsis plants with loss-of-function mutations in the IspE and IspF genes also have an albino phenotype (M.-H. Hsieh and H.M. Goodman, unpublished data). It seems that plants carrying a null mutation in any of the nonMVA pathway genes are albino lethal. Together, these results suggest that if isoprenoids synthesized via the cytosolic MVA pathway can be transported into chloroplasts, the amount used for phytol and carotenoid biosynthesis is insufficient for survival.

MATERIALS AND METHODS

Nomenclature

The nonMVA or MVA-independent pathway has also been called the DXP pathway or the MEP pathway in the literature (Eisenreich et al., 1998, 2001; Lichtenthaler, 1999; Rodriguez-Concepcion and Boronat, 2002). The nonMVA pathway enzymes are DXS; DXR or IspC; IspD (YgbP), CMS; IspE (YchB), CMK; IspF (YgbB), MCS; IspG (GcpE), HDS; IspH (LytB), HDR or IPP/DMAPP synthase (IDS).

Plant Material

Arabidopsis (Arabidopsis thaliana ecotype Columbia-0) was grown on half-strength Murashige and Skoog plates (Murashige and Skoog salts [GIBCO/BRL, Cleveland], pH adjusted to 5.7 with 1 n KOH, 0.8% [w/v] phytoagar) containing 2% Suc, or in soil in the greenhouse on a 16-h-light/8-h-dark cycle at 23°C. For experiments in which plants were transferred to 0% Suc, 3% Suc, or 3% mannitol (Fig. 9), seeds were sown on 1.5- × 8-cm nylon nets with 250-μm mesh size (Tetko, Elmsford, NY; catalog no. 3–250/50) placed on the surface of the media. For transfer to new media, the nylon nets were lifted, and the plants were transferred to fresh Murashige and Skoog media containing the indicated supplementations. Determination of total chlorophyll and carotenoids in three independent samples of 2-week-old Arabidopsis wild-type and ispH-1 seedlings grown in tissue culture was conducted as described (Linchtenhaler and Wellburn, 1983).

Isolation of Arabidopsis ispH-1 Mutants

A binary vector pBI121 with a kanamycin-selectable marker was transformed into Arabidopsis ecotype Columbia-0 to generate a collection of approximately 4,000 T-DNA lines. T2 seeds of each independent T-DNA line were used to screen for albino mutants. Thermal asymmetric interlaced-PCR was used to determine the T-DNA flanking genomic sequences of these mutants (Liu et al., 1995). In mutant line 3a234 (ispH-1), the albino phenotype cosegregates with a T-DNA insertion in the IspH locus.

Cloning of Arabidopsis IspH cDNA

Total RNA from 2-week-old Arabidopsis was used for reverse transcription-PCR (SuperScriptII reverse transcriptase kit; Invitrogen, Carlsbad, CA), and primers 5′-GTGCGTTTCTCTCGAACTCT-3′ and 5′-GGTAAGAACATTAAGTGGAG-3′ were used to amplify a full-length IspH cDNA. The PCR product was cloned into pCR2.1-TOPO (Invitrogen) and provided for sequencing. The Arabidopsis IspH cDNA sequence and the deduced amino acid sequence were deposited in GenBank (AY168881).

Complementation of the ispH-1 Mutant and Generation of IspH Gene-Silencing Lines

The full-length IspH cDNA driven by a cauliflower mosaic virus 35S promoter in the sense orientation was subcloned into a plant expression vector pSMAB704 containing the basta resistance (BAR) selectable marker and transformed by floral dip (Clough and Bent, 1998) into ispH-1 heterozygous (±) plants, for complementation testing, or into wild-type Arabidopsis plants for overexpression studies. In the complementation test, genomic DNA extracted from green BARR primary transformants was used to determine the genotype of the IspH locus (± or −/−) by genomic Southern analysis. Two of the 16 green BARR primary transformants tested were ispH (−/−) homozygous. In the T2 generation, more than 100 seeds from each of the 16 lines were germinated on a BAR selective medium and all BARR seedlings were green, an indication that the 35S:IspH transgene complements the albino phenotype in all 16 lines. Two ispH (−/−) homozygous lines (confirmed by genomic Southern analysis) were carried to T3 and were homozygous for the 35S:IspH transgene. Progeny of these two lines are all green when grown on regular Murashige and Skoog medium or on a selective medium containing kanamycin or phosphinothricin. In the overexpression experiment, 186 BARR primary transformants were obtained. Sixty-three of the 186 lines showed IspH gene silencing as indicated by pale green or various albino patterns. Of the 63 IspH-silencing plants, 30 died without setting seeds.

Northern-Blot Analysis

Arabidopsis total RNA was isolated using a phenol extraction protocol (Jackson and Larkins, 1976). For detection of IspH mRNA, a gene-specific digoxigenin (DIG)-labeled single-stranded DNA probe was generated by PCR using primers 5′-GTCGTGGAAGATGCTTTGGT-3′ and 5′-GGTAAGAACATTAAGTGGAG-3′ (Myerson, 1991). Primers 5′-GACAGACTGAGAGCTCTTTC-3′ and 5′-ACAGGTATCGACAATGATCC-3′ were used for making a DIG-labeled single-stranded DNA probe to detect the 18S rRNA. The following primers were used for making DIG-labeled gene-specific probes: DXS (U27099), 5′-TTGAGAGTAAGAATCTGTTG-3′, 5′-AGCTTATTGAAGATCACAAG-3′; DXR (AF148852), 5′-CTCTGATGATGACATTAAAC-3′, 5′-CAACCAATTCTTCATGCATG-3′; IspD (AF230737), 5′-ATGGCGATGCTTCAGACGAA-3′, 5′-TCATGAGTCCTCGCTCAAGA-3′; IspE (AF288615), 5′-ATGGCAACGGCTTCTCCTCC-3′, 5′-TCATTGGAAATCCATGCGAG-3′; IspF (AF321531), 5′-ATGGCTACTTCTTCTACTCA-3′, 5′-CTATTTCTTCATGAGGAGAA-3′; IspG (AY081261), 5′-ATGGCGACTGGAGTATTGCC-3′, 5′-CTACTCATCAGCCACGGGCG-3′; ASN1 (L29083), 5′-AACTCCGATAGCGGCTC-3′, 5′-CTCTATTTCCACAAGGCACC-3′; RBCS (X13611), 5′-GCTTCCCTTGTTCGGTTGCA-3′, 5′-CCGATAGAATATGTCTCGCA-3′. DIG probe labeling, prehybridization, hybridization, wash conditions, and detection were performed according to the Boehringer Mannheim Genius System User's Guide, version 3.0. All northern blots presented herein were repeated at least two times with comparable results.

Transmission Electron Microscopy

The leaf samples were fixed in 4% glutaraldehyde, 100 mm sodium cacodylate, pH 7.2, for 16 h at 4°C, and postfixed with 1% osmium tetroxide in the same buffer for 6 h at 4°C. The fixed samples were dehydrated through a series of alcohol solutions and embedded in Spurr resin. Ultrathin sections were cut on a Reichert Ultracut-S (Leica Microsystems, Bannockburn, IL) and stained with uranyl acetate and lead citrate and viewed with a transmission electron microscope, JEOL 1200EX (JEOL USA, Peabody, MA).

Antibody Preparation and Immunoblot Analysis

The Arabidopsis IspH cDNA was digested with SacI and BamHI and cloned into similarly cut pQE-30 (Qiagen, Valencia, CA) to express His-tagged IspH protein in Escherichia coli. The resulting clone, pQE-AtIspH, encodes a nearly complete mature Arabidopsis IspH protein missing the first 24 amino acid residues with a 6× His tag at the N terminus. Purification of the His-tagged IspH protein was performed according to The QIAexpressionist, fourth edition (Qiagen). Polyclonal antibodies were raised by immunization of a rabbit using the purified fusion protein (Cocalico Biologicals, Reamstown, PA). Total protein extraction, chloroplast isolation, and immunoblot analysis were performed as described (Hsieh et al., 1998). Total proteins were extracted from 3-week-old Arabidopsis seedlings. Intact chloroplasts isolated from 3-week-old Arabidopsis leaves were lysed and separated by centrifugation into soluble (stroma) and insoluble (membranes) fractions. The ECL+ system was used for detection in the immunoblot analysis (Amersham, Piscataway, NJ).

Complementation of the E. coli ispH Mutant

The E. coli ispH mutant strain MG1655 ara<>ispH was maintained on Luria-Bertani medium containing 50 μg/mL kanamycin and 0.2% (w/v) Ara (McAteer et al., 2001). The pQE-AtIspH plasmid was transformed into the E. coli ispH mutant and selected on Luria-Bertani plates containing 50 μg/mL kanamycin, 50 μg/mL ampicillin, 0.2% Glc, and 0.5 mm IPTG. The presence of the pQE-AtIspH plasmid in surviving colonies was verified. As a control, the empty pQE-30 vector was transformed into the E. coli ispH mutant and selected on Luria-Bertani plates containing 50 μg/mL kanamycin, 50 μg/mL ampicillin, and 0.2% Ara. The transformants containing the pQE-30 empty vector cannot grow on medium containing 0.2% Glc (Fig. 8, bottom right).

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY168881.

Acknowledgments

We thank Dr. J. Sheen for the pSMAB704 binary vector and OE33, LHCII, and rbcS polyclonal antibodies, and Dr. M. Masters for the E. coli ispH mutant strain.

Footnotes

  • Received December 21, 2004.
  • Revised February 26, 2005.
  • Accepted February 27, 2005.
  • Published April 29, 2005.

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The Arabidopsis IspH Homolog Is Involved in the Plastid Nonmevalonate Pathway of Isoprenoid Biosynthesis
Ming-Hsiun Hsieh, Howard M. Goodman
Plant Physiology Jun 2005, 138 (2) 641-653; DOI: 10.1104/pp.104.058735
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