The Arabidopsis IspH Homolog Is Involved in the Plastid ... · The Arabidopsis IspH Homolog Is Involved in the Plastid Nonmevalonate Pathway of Isoprenoid Biosynthesis Ming-Hsiun

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

Ming-Hsiun Hsieh* and Howard M. Goodman

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 (M.H.H., H.M.G.);Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114(M.H.H., H.M.G.); and Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529,Taiwan (M.H.H.)

Plant isoprenoids are synthesized via two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastidnonmevalonate pathway. The Escherichia coli IspH (LytB) protein is involved in the last step of the nonmevalonate pathway. Wehave isolated an Arabidopsis (Arabidopsis thaliana) ispH null mutant that has an albino phenotype and have generatedArabidopsis transgenic lines showing various albino patterns caused by IspH transgene-induced gene silencing. The initiationof albino phenotypes rendered by IspH gene silencing can arise independently from multiple sites of the same plant. Aftera spontaneous initiation, the albino phenotype is systemically spread toward younger tissues along the source-to-sink flowrelative to the initiation site. The development of chloroplasts is severely impaired in the IspH-deficient albino tissues. Insteadof thylakoids, mutant chloroplasts are filled with vesicles. Immunoblot analysis reveals that Arabidopsis IspH is a chloroplaststromal protein. Expression of Arabidopsis IspH complements the lethal phenotype of an E. coli ispH mutant. In 2-week-oldArabidopsis seedlings, the expression of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphatereductoisomerase (DXR), IspD, IspE, IspF, and IspG genes is induced by light, whereas the expression of the IspH gene isconstitutive. The addition of 3% sucrose in the media slightly increased levels of DXS, DXR, IspD, IspE, and IspF mRNA in thedark. In a 16-h-light/8-h-dark photoperiod, the accumulation of the IspH transcript oscillates with the highest levels detected inthe 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 ofIspH, 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 prod-ucts found in living organisms. Among the importantisoprenoids are compounds such as steroid hormonesin mammals, carotenoids and chlorophylls in plants,and ubiquinone or menaquinone in bacteria. Stillothers are medically important for human health,e.g. vitamins, hormones, and anticancer agents suchas Taxol (Sacchettini and Poulter, 1997).All isoprenoids are derived from a basic five-carbon

unit, isopentenyldiphosphate (IPP), and its allyl isomerdimethylallyl diphosphate (DMAPP). For decades, themevalonate (MVA) pathway was believed to be theonly route to synthesize IPP and DMAPP. However,recent studies have uncovered an alternative nonme-valonate (nonMVA) pathway for isoprenoid biosyn-thesis (Rohmer et al., 1993; Eisenreich et al., 1998, 2001;Lichtenthaler, 1999; Rohdich et al., 2001; Rodriguez-Concepcion and Boronat, 2002; Rohmer, 2003). Most, ifnot all, enzymes involved in the nonMVA pathwayhave been identified in Escherichia coli (Sprenger et al.,1997; Lois et al., 1998; Takahashi et al., 1998; Rohdichet al., 1999, 2002, 2003; Herz et al., 2000; Luttgen et al.,2000; Hecht et al., 2001; Adam et al., 2002). In the firststep, 1-deoxy-D-xylulose 5-phosphate synthase (DXS)

converts pyruvate and glyceraldehyde-3-phosphate to1-deoxy-D-xylulose 5-phosphate (DXP), which alsoserves as a biosynthetic precursor of vitamins B1(thiamine) and B6 (pyridoxal; White, 1978; Sprengeret al., 1997). DXP is converted to 2C-methyl-D-erythritol4-phosphate (MEP) by the 1-deoxy-D-xylulose 5-phos-phate reductoisomerase (DXR or IspC). MEP isthen converted to IPP and DMAPP in consecutivesteps catalyzed by 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS or IspD), 4-diphosphocy-tidyl-2-C-methyl-D-erythritol kinase (CMK or IspE),2-C-methyl-D-erythritol 2,4-cyclodiphosphate syn-thase (MCS or IspF), 1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase (HDSor IspG), and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDRor IspH; Fig. 1). Because MEP is the first committedprecursor in the pathway, the nonMVApathway is alsoknown as the MEP pathway.

The nonMVA pathway has been found in a broadrange of organisms, including bacteria, green algae,and higher plants (Eisenreich et al., 1998, 2001;Lichtenthaler, 1999; Cunningham et al., 2000; Rohdichet al., 2001; Rodriguez-Concepcion and Boronat, 2002).In plants, the MVA and nonMVA pathways are com-partmentalized in the cytoplasm and plastid, respec-tively. Sesquiterpenes, sterols, and polyterpenes arederived from the cytosolic MVA pathway, whereas iso-prene, phytol, carotenoids, and plant hormones GAand abscisic acid are synthesized via the plastid non-MVA pathway (Fig. 1). The Arabidopsis genome

* Corresponding author; e-mail [email protected]; fax886–2–2782–7954.

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.104.058735.

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contains genes encoding homologs of E. coli nonMVApathway enzymes and the deduced amino acidsequences all possess a transit peptide for chloroplastlocalization, consistent with their predicted role inthe biosynthesis of plastid isoprenoids (Rodriguez-Concepcion and Boronat, 2002).

Previous studies have shown that Arabidopsisplants (cla1-1 mutants) with a null mutation in theDXS gene are albino (Mandel et al., 1996; Estevez et al.,2000, 2001). Analyses of the flanking genomic DNAsequences of a collection of Arabidopsis T-DNA ortransposon-tagged seedling lethal lines have identifiedalbino mutants in the DXS, IspC (DXR), and IspDgenes, although these lines have not been furthercharacterized (Budziszewski et al., 2001). Levels ofphotosynthetic pigments are dramatically reduced inthe Arabidopsis IspD antisense lines (Okada et al.,2002). The albino phenotype was also observed inNicotiana 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 revealedthat the clb4 and clb6 mutants are caused by the lossof function of the IspG and IspH genes, respectively(Gutierrez-Nava et al., 2004; Guevara-Garcia et al.,2005). These observations suggest that plants carry-ing loss-of-function mutations in any of the nonMVApathway genes may have a visible pigmentationphenotype.

To isolate plant nonMVA pathway mutants, wegenerated Arabidopsis T-DNA insertion lines andscreened for plants showing pale green or albinophenotypes. One of the isolated Arabidopsis albinomutants is caused by a T-DNA insertion in a gene thatencodes a protein with significant similarity to E. coliIspH (or LytB). Consistent with the albino phenotypeobserved in the null mutant, Arabidopsis IspH gene-silencing plants show pale green to various albinopatterns. Levels of IspH mRNA are dramatically re-duced in the IspH-silenced albino tissues. We alsoprovide experimental evidence that the ArabidopsisIspH protein is localized in the chloroplast stroma. Acomplementation test with an E. coli ispH mutantfurther confirms that the Arabidopsis IspH proteinfunctions as a nonMVA pathway enzyme involved inthe biosynthesis of plastid isoprenoids.

The biosynthesis of plastid isoprenoids is directlylinked to photosynthesis. We have thus examined theeffects of light and Suc on the expression of nonMVApathway genes in Arabidopsis. In addition, it has beensuggested that the biosynthesis and emission of vola-tile plant isoprenoids are derived from the plastidnonMVA pathway (Lichtenthaler, 1999; Sharkey andYeh, 2001; Zeidler and Lichtenthaler, 2001). The emis-sion of some volatile plant isoprenoids and the ex-pression of some terpene synthase genes are regulateddiurnally or nocturnally (Loreto et al., 1996; Kolosovaet al., 2001; Lu et al., 2002; Dudareva et al., 2003; Martinet al., 2003). We also studied the expression patternsof Arabidopsis nonMVA pathway genes under a 16-h-light/8-h-dark photoperiod. Several distinct diurnalexpression patterns were observed among the Arabi-dopsis nonMVA pathway genes. The accumulation ofDXS, IspG, and IspH transcripts oscillates in a similarpattern during the 16-h-light/8-h-dark cycle.

RESULTS

Phenotypic Analysis of the ArabidopsisispH-1 Mutant

We isolated the albino ispH-1 mutant by screeninga collection of Arabidopsis T-DNA insertion lines.Genetic analysis and thermal asymmetric interlaced-PCR revealed that the albino line 3a234 containstwo copies of T-DNA in two different loci, IspH andAt3g46440, which were further segregated as twodifferent lines. Homozygous ispH-1 plants are albinoand progeny from a self-pollinated heterozygous plantsegregate green and albino plants in a 3:1 ratio ona nonselective medium, i.e. the albino phenotype is

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-diphosphocy-tidyl-2-C-methyl-D-erythritol; CDP-ME2P, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; ME-2,4cPP, 2-C-methyl-D-eryth-ritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate; GGPP, geranylgeranyl diphosphate; GA, gibberellicacid; PQ, plastoquinone; ABA, abscisic acid. Enzymes of theMVA path-way: 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, DOXPreductoisomerase; CMS, CDP-ME synthase; CMK, CDP-ME kinase;MCS, ME-2,4cPP synthase; HDS, HMBPP synthase; HDR, HMBPPreductase. The names of their corresponding genes are indicated on theleft.

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inherited as a recessive mutation (Fig. 2A). The ispH-1mutant seedlings exhibit a purple-tinted phenotypesuperimposed on the albino phenotype when grownon the medium containing Suc (Fig. 2A). The purplecoloration begins to fade about 1 week after germi-nation on this medium. The ispH-1 albino plant candevelop a normal root system, rosette leaves, an in-florescence with cauline leaves, and flower-like struc-tures that never mature into normal flowers whengrown on tissue culture medium (Fig. 2B).To study the effect of the ispH-1 mutation on

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

Molecular Characterization and Complementation

of the ispH-1 Locus

Analysis of the flanking genomic DNA sequencesrevealed that the Arabidopsis ispH-1 mutant has aT-DNA insertion in the seventh exon of the IspH gene(Fig. 3A). Northern and immunoblot analyses showedthat the IspH mRNA and protein were undetectable inthe ispH-1 mutant (Fig. 3B). These results suggest thatispH-1 is a null mutant. In 6-week-old wild-type Arab-idopsis plants, the IspH transcript was detected in all

tissues analyzed (Fig. 3C). To prove that the defectiveispH-1 locus is responsible for the albino phenotype,werestored the wild-type phenotype by introducing intothe mutant a full-length IspH cDNA transcribed froma cauliflower mosaic virus 35S promoter. The pheno-type of a representative complementation line is shownin Figure 3D. Genomic DNA-blot analysis was usedto verify that the complemented plants containeda (homozygous) ispH-1 mutant allele and a 35S:IspHtransgene (Fig. 3E). These results confirm that thealbino phenotype is caused by disruption of the IspHgene.

Arabidopsis 35S:IspH cDNA Transgene-InducedGene Silencing

Attempts to create Arabidopsis IspH overexpressionlines resulted in some primary transformants showingpale green or various albino phenotypes (Fig. 4, A andB). In plants, some transgenesmay cause a coordinatedsilencing of the transgene and homologous host genes(Mlotshwa et al., 2002). It is possible that the IspH geneis silenced in the albino tissue. To test this, total RNAand protein extracted from green and albino tissues ofthe transgenic plants were examined by northern andimmunoblot analysis. The steady-state levels of IspHmRNA and protein are higher in the transgenic greentissue than in the wild type, whereas the IspH mRNAand protein are not detectable in the albino tissue (Fig.4, C and D). These results indicate that IspH is over-expressed in the green tissue and is silenced in thealbino tissue of the same plant.

Initiation and Systemic Spread of IspHGene Silencing

The visual albino phenotype that is a result of IspHgene silencing serves as a marker for observing theinitiation and systemic spread of transgene-inducedgene silencing in Arabidopsis. The initiation of IspHgene silencing is spontaneous and stochastic; it mayarise at various developmental stages and severalindependent initiations may even occur in the sameplant. For instance, the albino phenotype may appearindependently in rosette leaves, stems, and siliques(Fig. 5A). After the initiation step, somehow the IspHgene-silencing signal(s) is systemically spread towarddeveloping tissues so that younger tissues that de-velop above the initiation site will be affected (Fig. 5,A–C). Expanding cauline leaves, at the time of initia-tion, either are not or are only partially affected,leading to a phenotype where a green leaf and a

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

Table I. Photosynthetic pigment content of wild-type Arabidopsisand ispH-1 mutants

Values shown are mg/g fresh weight 6 SE.

Chlorophyll a Chlorophyll b Carotenoids

Wild type 577.6 6 1.3 271.1 6 10.3 159.7 6 3.2ispH-1 1.1 6 0.8 2.6 6 0.5 3.2 6 0.8

Arabidopsis ispH Mutant

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partially green leaf are attached to an albino stem(Fig. 5D). Sometimes the apical region of a silencedinflorescence may remain green, which indicates thatthe IspH gene is not always silenced in the meriste-matic regions (Fig. 5, A, D, and L). In siliques, IspHgene silencing can be localized in the base, in the tip, inthe middle, or at both ends independently and grad-ually spreads throughout the entire silique (Fig. 5, A,D, and E). The random initiation and systemic spreadof the albino phenotype in siliques indicate that cells indeveloping 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 non-silenced (green) and silenced plants with various al-bino patterns segregate randomly (Fig. 5F).

When the initiation of IspH gene silencing is localizedin rosette leaves during the vegetative stage, leaves thathave 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 reproductivestage, the green inflorescence tip has a chance to de-velop flowers and siliques before the entire inflores-cence becomes albino (Fig. 5, J–L).Neither initiationnorsystemic spread of IspH gene silencingwas observed infully expanded rosette leaves.

Dynamics of Thylakoids in the IspH-Silencing Tissues

In a partially silenced rosette leaf, chloroplasts in thegreen cells accumulate more starch granules than thecomparable wild type (Fig. 6, A and B), whereas inthe IspH-silenced albino tissue, chloroplasts are highlyvesiculated (Fig. 6C). Because the basal part of anexpanding leaf is composed of younger tissues, it ispossible that these vesicles are derived from undif-ferentiated chloroplasts. During the systemic spread ofIspH gene silencing in a leaf, a narrow boundary line ofpale green to pale yellow forms between the green andthe albino tissue. Transmission electron microscopyreveals that various types of chloroplasts exist in thisregion (Fig. 6, D–I). Chloroplasts of pale green tissuesclose to the nonsilenced green part of the leafhave highly differentiated thylakoids, but most of thestroma lamellae are discontinuous and stacked thy-lakoids are thicker than the wild type (Fig. 6D). Bycontrast, chloroplasts of pale yellow tissues close tothe albino part of the leaf have only a few differenti-ated thylakoids (Fig. 6, E and F), mixed vesicles andloosening thylakoids (Fig. 6G), small vesicles (Fig. 6H),or large vesicles (Fig. 6I). The IspH-silencing chloro-plasts also contain densely stained globule (lipid-droplet) aggregates (Fig. 6, D, E, G, and I). Since thesystemic spread of the albino phenotype starts fromthe initiation site toward developing tissues, thesechloroplasts may represent a broad range of undiffer-entiated, partially differentiated, and fully differenti-ated chloroplasts that are affected by photooxidationcaused by various levels of IspH gene silencing. Thevesicular structures and densely stained globule

Figure 3. A, Schematic diagram of the Arabidopsis IspH gene. Arrowsindicate EcoRV restriction sites. Black boxes indicate exons. TheT-DNA (white triangle) is not drawn to scale. B, Northern and immuno-blot analyses. Total RNA (10 mg) and proteins (20 mg) extracted from2-week-old wild-type (WT) and ispH-1 plants were used for northern(top) and immunoblot (bottom) analyses to detect the IspH mRNA andIspH protein, respectively. After detection of the IspH mRNA, themembrane was stripped and reprobed with 18S rDNA as a control(middle). C, Expression pattern of the Arabidopsis IspH gene. Total RNA(10 mg) extracted from 6-week-old wild-type Arabidopsis plants grownin soil was used for northern-blot analysis. R, Roots; L, leaves; St, stems;F, flowers; Si, siliques. The ethidium bromide-stained agarose gel of thesame samples is shown at the bottom. D, Eight-day-old Arabidopsiswild-type (WT), ispH-1, and 35S:IspH cDNA complemented (Com)seedlings. E, Genomic Southern analysis (EcoRV digested). The arrowindicates the ispH-1 mutant allele and the arrowhead indicates the35S:IspH transgenic allele in a complemented (Com) line.

Figure 4. Arabidopsis 35S:IspH cDNA transgene-induced gene silenc-ing. A, Schematic diagram of a 35S:IspH cDNA construct. B, Repre-sentative primary transformants of 35S:IspH Arabidopsis after BASTAtreatment. Red arrows indicate IspH-silencing plants. C, Northern-blotanalysis 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.

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aggregates observed in these chloroplasts may bederived from the precursor components or breakdownproducts of thylakoids.

Arabidopsis IspH Is a Chloroplast Stromal Protein

Alignment of IspHamino acid sequences fromplants(Arabidopsis and Adonis), cyanobacterium, and E. coliindicates that both plant IspH proteins have an extraN-terminal sequence with the features of a chloroplasttransit peptide (data not shown; Botella-Pavia et al.,2004). Chloroplast fractionation and immunoblot anal-ysis showed that the Arabidopsis IspH protein isassociated with the soluble fraction (Fig. 7), which isindicative of localization in the stroma. Control assayswere performed to detect the membrane-localizedOE33 and light-harvesting complex (LHC)-II proteinsand the soluble, stroma-localized small subunit ofRubisco (rbcS) in the same samples (Fig. 7).

Arabidopsis IspH Complements theE. coli ispH Mutant

To test whether the Arabidopsis IspH protein hassimilar enzymatic activity to its E. coli counterpart, we

performed a complementation assay with an E. coliispH mutant. In E. coli ispH mutant strain MG1655ara,.ispH, the endogenous IspH gene was replacedby a kanamycin-resistant cassette and a single copyof IspH was present on the chromosome under thecontrol 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 ofarabinose (Ara; Fig. 8, left). Growth of the E. coliMG1655 ara,.ispH mutant on the medium contain-ing Glc was restored successfully by transformation ofthe pQE-AtIspH plasmid but not by the empty pQE-30vector (Fig. 8, right).

Light Induction of the nonMVA Pathway Genes

It has been shown that expression of ArabidopsisDXS andDXR (IspC) genes is induced by light (Mandelet al., 1996; Carretero-Paulet et al., 2002). We thusexamined the effects of light/dark and Suc on theexpression of Arabidopsis IspH and other nonMVApathway genes. Two-week-old Arabidopsis seedlingsgrown under normal light conditions (16-h-light/8-h-dark cycle) were subsequently placed in continuouslight or dark for 48 h. During the 48-h dark or light

Figure 5. Systemic spread of IspH genesilencing in Arabidopsis. A, Initiation andsubsequent systemic spread of IspH genesilencing occurred in rosette leaves, stems(primary and lateral), and siliques inde-pendently. B, Pale green phenotype of anIspH-silenced plant. Progeny derived fromthis plant segregate green, pale green, andvarious albino patterns randomly. C, Al-bino inflorescence of an IspH-silencedplant. D, Green and partially green cau-line leaves attached to an IspH-silenced(albino) stem. The arrow indicates thealbino tip of an IspH-silenced silique. E,Wild-type (left) and various albino pheno-types of IspH-silenced siliques. F, Fourrepresentative T3 homozygous lines. Lines1, 3, and 4 randomly segregate silenced(with various albino patterns) and non-silenced (green) plants. All plants in line 2are green. G to I, Systemic spread of thealbino phenotype toward developing ro-sette leaves. The plant shown in G, 3 d (H)and 6 d (I) later. J, Initiation of IspH genesilencing in the base (indicated by anarrow) of an expanding rosette leaf. Theplant shown in J, 3 d (K) and 9 d (L) later.





treatments, plants were grown in media containing 0%Suc, 3% Suc, or 3% mannitol. The nonmetabolizablesugar mannitol was included as an osmotic control.Total RNA extracted from these samples was used fornorthern-blot analysis to detect the steady-statemRNAlevels of the nonMVApathway genes. Compared to thedark treatments, light significantly increased the levelsofDXS,DXR, IspD, IspE, IspF, and IspGmRNAs (Fig. 9).In the dark, the addition of 3% Suc in themedia slightlyincreased the accumulation of DXS, DXR, IspD, IspE,and IspF transcripts (Fig. 9; compare 0% and 3% Suc inthe dark). This Suc induction is not due to an osmoticchange because the treatment with mannitol has nosignificant effect on levels ofDXS,DXR, IspD, IspE, and

IspF mRNAs (Fig. 9; compare 0% and 3% mannitol inthe dark). In contrast to the rest of the nonMVApathway genes, the steady-state levels of IspH mRNAare relatively constant in response to treatments withlight/dark or Suc (Fig. 9).

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

To further investigate whether expression of thenonMVA pathway genes is coordinately regulated, wecompared the day/night expression patterns of thesegenes in 13- and 14-d-old Arabidopsis seedlings (Fig.10). Interestingly, several distinct diurnal expressionpatterns were observed in the Arabidopsis nonMVApathway genes. The expression patterns of DXS, IspG,and IspH are similar during the 16-h-light/8-h-darkcycle. Peak levels of DXS, IspG, and IspH mRNAweredetected in the early period of the light cycle (2–6 h inthe light) and in the late period of the dark cycle (6–8 hin the dark). An additional peak appeared at the end ofthe light cycle (16 h), which is more evident in relativemRNA levels ofDXS and IspG and less obvious in thatof IspH (Fig. 10B). Oscillations in DXR, IspD, IspE, andIspF mRNA accumulation also occurred during thelight/dark cycle. In contrast to DXS, IspG, and IspH,peak levels of IspD mRNA appeared in the late periodof the light cycle (12–16 h). The highest levels of DXR,IspE, and IspF mRNA were also detected in the lateperiod (14 h) of the light cycle. An additional peak inthe early period (2–6 h) of the light cycle is evidentin the relative mRNA levels of IspE and less obvious inthose of DXR and IspF. Interestingly, the expressionof all nonMVA pathway genes is significantly re-pressed during the transition from light to dark (Fig.10; compare light 16 h to dark 2 h in each cycle). Thesame RNA samples were used to detect the light/darkexpression patterns of Arabidopsis ASN1 (encodingAsn synthetase) and rbcS (encoding Rubisco smallsubunit) as controls. The expression of ArabidopsisASN1 is induced by dark and repressed by light (Lamet al., 1998). During a normal 16-h-light/8-h-dark

Figure 6. Transmission electron micrographs. Ultrastructure of chloro-plasts from (A) wild-type rosette leaves, (B) green, and (C) albino tissuesof an IspH-silencing leaf. D to I, Ultrastructure of chloroplasts froma pale green to pale yellow boundary between the green and albinotissue of an IspH-silencing leaf. Scale bars, 500 nm. A 5-week-oldArabidopsis IspH gene-silencing plant used for transmission electronmicroscopy is shown on the top.

Figure 7. Arabidopsis IspH protein is localized in the chloroplaststroma. Fifteen micrograms of total proteins (T), chloroplast stromalproteins (Chl.S), and chloroplast membrane proteins (Chl.M) were usedfor immunoblot analysis. After detection with the IspH antibody, themembrane was stripped and reprobedwith OE33 antibody to detect themembrane-localized protein. The same protein samples were analyzedin a replicate membrane to detect the stroma-localized rbcS and themembrane-localized LHC-II chlorophyll a/b-binding protein.

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cycle, peak levels of ASN1 mRNA appear in themiddle of the light cycle and in the dark cycle (Hsieh,1998). The expression of Arabidopsis rbcS is inducedby light (Dedonder et al., 1993) and oscillates duringa light/dark cycle (Pilgrim and McClung, 1993).

DISCUSSION

The E. coli LytB protein was originally identified asone of the components involved in penicillin toleranceand control of the stringent response (Gustafson et al.,1993). Studies on a lytB mutant of the cyanobacteriumSynechocystis strain PCC 6803 provided evidence ofa role for LytB in the nonMVA pathway of isoprenoidbiosynthesis (Cunningham et al., 2000). Mutagenesisstudies in E. coli also suggested that the LytB protein isinvolved in the nonMVA pathway (Altincicek et al.,2001; McAteer et al., 2001). Recent in vivo and in vitroexperiments have confirmed that E. coli LytB catalyzesthe last reaction of the nonMVA pathway, a branchingstep that separately produces IPP and DMAPP ina ratio of 5:1 to 6:1 (Adam et al., 2002; Altincicek et al.,2002; Rohdich et al., 2002, 2003). E. coli LytB was thusrenamed IspH (Adam et al., 2002).Here, we report the identification and characteriza-

tion of an Arabidopsis albino mutant that has a nullmutation in the IspH gene. The phenotype of the ispH-1mutant and the IspH gene-silencing lines and thelocalization of the IspH protein are in accord witha role for Arabidopsis IspH in plastid isoprenoid bio-synthesis. Consistentwith our studies, Page et al. (2004)have shown that leaves ofN. benthamianawith the TRV-silenced IspH gene also have an albino phenotype. Theclbmutant clb6-1 carries a mutation in the ArabidopsisIspH gene (Gutierrez-Nava et al., 2004; Guevara-Garciaet al., 2005). The plastid nonMVA pathway is the

primary contributor for the biosynthesis of chloro-phylls and carotenoids, which are essential compo-nents for chloroplast development andplant growth. InIspH-deficient albino cells, the biosynthesis of photo-synthetic pigments is impaired and, thus, thylakoidscannot fully develop, which, in turn, results in an accu-mulation of large vesicles inside the mutant chloro-plast.

The phenomenon of transgene-induced gene silenc-ing was first uncovered as coordinate silencing (cosup-pression) of both the transgene and the homologousplant gene in petunia (Napoli et al., 1990; Van der Krolet al., 1990). It was later shown to occur at the post-transcriptional level (De Carvalho et al., 1992; VanBlokland et al., 1994). Posttranscriptional gene silenc-ing (PTGS) in plants is mechanistically similar to RNAinterference in animals (Fire et al., 1998). Despite beingavidly studied in recent years, the epigenetic mecha-nisms of the initiation, propagation, and maintenanceof transgene-induced gene silencing are still largelyunknown. It has been suggested that the initiation ofPTGS could correspond to highly transcribed single-transgene copies ordependon the transgeneproducinga particular form of RNA above a threshold level(Vaucheret et al., 2001). Because the expression of thetransgene or the accumulation of the particular formof RNA varies in each cell, the initiation of PTGSin transgenic plants is stochastic and localized. Our

Figure 8. Arabidopsis IspH complements the E. coli ispH mutant. TheE. coli ispH mutant strain MG1655 ara,.IspH was able to grow onLuria-Bertani media containing 0.2% Ara, but not on media containing0.2% Glc (left). After transformation with the Arabidopsis IspH cDNA(pQE-AtIspH) and, as a control, with the empty vector (pQE) alone, theresulting strains were tested for growth on media containing 0.2% Glc(right).

Figure 9. Northern-blot analysis of Arabidopsis nonMVA pathwaygenes. Total RNA (10 mg) extracted from 2-week-old Arabidopsisseedlings treated with light or dark for 48 h in the presence or absenceof 3% Suc was used to detect the expression of nonMVA pathwaygenes. Steady-state levels of DXS, DXR, IspD, IspE, IspF, and IspGmRNAs are significantly increased by light. Suc, Sucrose; Man,mannitol. The ethidium bromide-stained agarose gel of the samesamples is shown at the bottom.





Figure 10. Expression patterns of Arabidopsis nonMVA pathway genes in a normal day/night cycle. A, Arabidopsis seedlingswere grown on tissue culture plates (Murashige and Skoog plus 3% Suc) in a 16-h-light/8-h-dark cycle, and samples werecollected every 2 h on days 13 and 14. Total RNA (10 mg) extracted from these samples was used to detect the expression patternsof the nonMVA pathway genes. The light/dark expression patterns of Arabidopsis ASN1 and rbcSwere also detected in the sameRNA 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 signalswere 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 softwarerepresent the relative mRNA levels of the nonMVA pathway genes, ASN1, and rbcS in A.

Hsieh and Goodman




observations on the initiation of Arabidopsis IspH genesilencing further support the notion that it may occurspontaneously. For instance, multiple initiation sitesmay arise independently in the same transgenic plant(Fig. 5A). Regardless of the same genotype in the IspHtransgenic T3 homozygous lines, silenced (albino) andnonsilenced (green) progeny still segregate randomly(Fig. 5F).Moreover, the locations of initiation sites in theIspH-silenced lines vary from plant to plant and areunpredictable. These variations in triggering IspH genesilencing may reflect the different expression levels ofthe IspH transgene or the variable abundance of a par-ticular form of IspH mRNA in each cell.The systemic spread of the IspH-silenced albino

phenotype toward younger developing tissues, in-cluding the apical meristem (Fig. 5, A, C, and G–L),suggests that the transmission of IspH-silencing sig-nals is unidirectional and of high efficiency. In addi-tion, there is no obvious temporal difference in theappearance of albino phenotypes between vein andnonvein tissues during the spread of IspH gene silenc-ing (Fig. 5, G–L). The resulting IspH-silenced tissuesare uniformly photobleached rather than variegated.The direction of systemic spread of IspH gene silencingparallels the flow of metabolic source to sink in leavesand stems. In siliques, however, the spread of silencingsignals is stochastic (Fig. 5E). The processes involvedin carpel and fruit development are not well under-stood in Arabidopsis (Ferrandiz et al., 1999). It is likelythat the metabolic status (e.g. source versus sink)between the tip and the base of siliques is not asdistinct as those in leaves. In plants, several lines ofevidence indicate that the cell-to-cell systemic spreadof PTGS occurs through plasmodesmata and phloem(Palauqui et al., 1997; Jorgensen et al., 1998; Voinnetet 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- to25-nucleotide small interfering RNAs have been con-sidered as likely candidates for the systemic silencingsignal (Hamilton and Baulcombe, 1999; Himber et al.,2003; Yoo et al., 2004). The recently identified pumpkin(Cucurbita maxima) phloem SMALL RNA BINDINGPROTEIN1 (CmPSRP1) has been proposed to be in-volved in small RNA trafficking (Yoo et al., 2004). Atransmembrane protein SID-1 (systemic RNA interfer-ence deficient) required for systemic RNA silencinghas been identified in Caenorhabditis elegans (Winstonet al., 2002; Feinberg and Hunter, 2003). However,there are no apparent homologs of CmPSRP1 andSID-1 in the Arabidopsis genome. Further studies onIspH gene-silencing lines may help uncover the molec-ular components involved in the initiation and sys-temic spread of gene silencing in Arabidopsis.Taking advantage of the systemic spread of

the albino phenotype from the initiation site towarddeveloping tissues, we have observed a series ofmorphological changes in the chloroplasts of IspHgene-silencing cells (Fig. 6, D–I). The integration of the

LHCs, which are mainly composed of chlorophylls,carotenoids, and apoproteins, is important for thedevelopment of thylakoids and the formation of granastacking (Bartley and Scolnik, 1995; Von Wettsteinet al., 1995; Simidjiev et al., 2000). Thus, the develop-ment of the chloroplast in the IspH-silencing cells maybe arrested at various stages depending on the levelsof silencing. Because thylakoids are mainly composedof galactolipids containing highly unsaturated fattyacids, chloroplast membranes are very sensitive tophotooxidative damage (Dormann et al., 1999;Vothknecht and Westhoff, 2001). Carotenoids can pro-tect the photosynthetic apparatus from photooxidativedamage (Bartley and Scolnik, 1995; Niyogi, 1999). Withthe gradual loss of carotenoids in the IspH gene-silencing tissues, thylakoids may lose this protectionunder light and suffer various levels of photooxidativedamage. Since carotenoids have essential functions inphotosynthesis and photoprotection, those chloro-plasts observed in IspH-silencing tissues may notrepresent the different developmental stages of wild-type chloroplasts.

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

In addition to functional analysis and subcellularlocalization of the Arabidopsis IspH protein, we alsocharacterized the expression and regulation of theArabidopsis IspH gene. The Arabidopsis IspH tran-scripts are detected in all parts of adult plants, in-dicating that the IspH protein has an essential functionthroughout the entire plant (Fig. 3C). The expression ofthe Arabidopsis IspH gene in both photosynthetic andnonphotosynthetic tissues supports the notion that thenonMVApathway is involved in synthesizing a varietyof isoprenoids in plants. Consistent with their roles insynthesizing carotenoids and chlorophylls for photo-synthesis, the Arabidopsis nonMVA pathway genesare highly expressed in light and most are low in thedark (Fig. 9). The only exception is the IspH gene,whose expression is constitutive regardless of thecontinuous light/dark treatments in 2-week-old Arab-idopsis plants. Interestingly, it has been shown that theexpression of the IspH gene is up-regulated duringArabidopsis seedling deetiolation and levels of Arabi-dopsis IspH mRNA are significantly higher in 3-d-oldseedlings grown in continuous light than those ofdark-grown seedlings (Botella-Pavia et al., 2004). In





addition, Guevara-Garcia et al. (2005) have shown thatthe transcript levels of the Arabidopsis nonMVApathway genes are modulated during developmentwith their lowest levels detected in 3-d-old seedlings.Coordinated regulation of all Arabidopsis nonMVApathway genes only occurs during early developmen-tal stages (between 3 and 6 d) and the accumulationkinetics differ for each gene later in development(Guevara-Garcia et al., 2005). These results, togetherwith our studies, indicate that the expression of theArabidopsis IspH gene may be differentially regulatedby light during different developmental stages.

Since the initial substrates of the nonMVA pathway,pyruvate and glyceraldehyde 3-phosphate, may di-rectly derive from glycolysis and photosynthesis in thechloroplast, carbon metabolites may affect the expres-sion of the nonMVA pathway genes. We have foundthat the expression of the DXS, DXR, IspD, IspE, andIspF genes is slightly induced by Suc in the dark,whereas the presence of Suc in the light has noadditive effects beyond the light induction of thenonMVA pathway genes. These results suggest thatat least some of the Arabidopsis nonMVA pathwaygenes are subjected to metabolic regulation.

Although the expression of the IspH gene is notaffected 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, DXSand IspG share the most similar expression patternswith IspH during a normal light/dark cycle. Interest-ingly, expression of the Arabidopsis DXS and IspHgenes is coordinately regulated during deetiolation(Botella-Pavia et al., 2004). The first enzyme of thenonMVA pathway, DXS, has been proposed to bea limiting enzyme for the biosynthesis of plastidisoprenoids in Arabidopsis (Estevez et al., 2001).Studies on overexpression of tomato IspH cDNA inArabidopsis plants led to the conclusion that plantIspH protein also plays a key role in controlling thebiosynthesis of plastid isoprenoids (Botella-Pavia et al.,2004). Our studies show that expression of DXS andIspH is also coordinately regulated during a normallight/dark cycle in 2-week-old Arabidopsis plants.Consistent with these results, the diurnal expressionof Arabidopsis DXS and IspH genes has also beendetected by using microarray analysis (Harmer et al.,2000; Schaffer et al., 2001). Our observations of severaldistinct diurnal expression patterns of nonMVA path-way genes support the notion that some specificregulatory mechanisms may exist among these genesin 2-week-old Arabidopsis. It will be interesting tofurther test whether the expression of ArabidopsisnonMVA pathway genes is regulated by circadianrhythm. Recent studies by Guevara-Garcia et al. (2005)have shown that the nonMVA pathway is regulatedposttranscriptionally. Thus, it is important to investi-gate whether levels of the nonMVA proteins oscillatein similar patterns as their corresponding transcriptsduring a normal day/night cycle.

The MVA pathway in animal cells is regulated atmultiple levels, including transcriptional, posttran-scriptional, and a complex feedback regulatory system(Goldstein and Brown, 1990). Because the nonMVApathway genes have been uncovered just recently,little is known about their regulation in plants. It willbe very useful if we could develop techniques todirectly measure the activities of the nonMVA path-way enzymes in plants. Further studies on how theArabidopsis nonMVA pathway enzymes are regulatedand how the pathway is integrated into the upstreamand downstream pathways will provide insights intothe complex regulatory network of isoprenoid bio-synthesis in plants.

The existence of two independent IPP biosyntheticpathways inside a plant cell raises an interestingquestion as to whether and, if so, how these twopathways interact with each other. It has been sug-gested that interactions between the cytosolic MVApathway and the plastid nonMVA pathway may existin plants (Eisenreich et al., 1998, 2001; Lichtenthaler,1999; Kasahara et al., 2002; Nagata et al., 2002;Rodriguez-Concepcion and Boronat, 2002; Bick andLange, 2003; Laule et al., 2003; Rohmer, 2003). Thecross-flow of the compartmentalized isoprenoids maydepend on the plant species and has been estimated tobe less than 1% in intact plants under physiologicalconditions (Eisenreich et al., 2001). Recent studies byLaule et al. (2003) suggest that cross-talk betweenArabidopsis MVA and nonMVA pathways may occurmainly as posttranscriptional processes. Bick andLange (2003) have shown that the unidirectionaltransport of IPP and geranyl diphosphate from plas-tids to cytosol occurs in plants. The albino lethalphenotype of the ispH-1 mutant implies that the influxof cytosolic isoprenoids into chloroplasts does notoccur or, at best, is very limited in Arabidopsis. Thereported albino phenotype of Arabidopsis mutantswith disrupted DXS (i.e. cla1-1), DXR (IspC), IspD, andIspG (i.e. clb4) genes and the inhibitor fosmidomycin-treated plants also support this notion (Mandelet 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 alsohave an albino phenotype (M.-H. Hsieh and H.M.Goodman, unpublished data). It seems that plantscarrying a null mutation in any of the nonMVApathway genes are albino lethal. Together, these re-sults suggest that if isoprenoids synthesized via thecytosolic MVA pathway can be transported into chlor-oplasts, the amount used for phytol and carotenoidbiosynthesis 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

Hsieh and Goodman




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- 3 8-cm nylon nets with

250-mm mesh size (Tetko, Elmsford, NY; catalog no. 3–250/50) placed on

the surface of the media. For transfer to newmedia, the nylon nets were lifted,

and the plants were transferred to fresh Murashige and Skoog media contain-

ing 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 trans-

formed 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 transcrip-

tion-PCR (SuperScriptII reverse transcriptase kit; Invitrogen, Carlsbad, CA),

and primers 5#-GTGCGTTTCTCTCGAACTCT-3# and 5#-GGTAAGAACAT-

TAAGTGGAG-3# were used to amplify a full-length IspH cDNA. The PCR

product was cloned into pCR2.1-TOPO (Invitrogen) and provided for se-

quencing. The Arabidopsis IspH cDNA sequence and the deduced amino acid

sequence were deposited in GenBank (AY168881).

Complementation of the ispH-1 Mutant and Generationof 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

(6) 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 (6 or2/2) by genomic Southern analysis. Two

of the 16 green BARR primary transformants tested were ispH (2/2)

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 (2/2) 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#-GGTAAGAAC-

ATTAAGTGGAG-3# (Myerson, 1991). Primers 5#-GACAGACTGAGAGCT-

CTTTC-3# and 5#-ACAGGTATCGACAATGATCC-3# were used for making

a DIG-labeled single-stranded DNA probe to detect the 18S rRNA. The follow-

ing primers were used for making DIG-labeled gene-specific probes: DXS

(U27099), 5#-TTGAGAGTAAGAATCTGTTG-3#, 5#-AGCTTATTGAAGATCA-

CAAG-3#; DXR (AF148852), 5#-CTCTGATGATGACATTAAAC-3#, 5#-CAA-

CCAATTCTTCATGCATG-3#; IspD (AF230737), 5#-ATGGCGATGCTTCA-

GACGAA-3#, 5#-TCATGAGTCCTCGCTCAAGA-3#; IspE (AF288615),

5#-ATGGCAACGGCTTCTCCTCC-3#, 5#-TCATTGGAAATCCATGCGAG-3#;IspF (AF321531), 5#-ATGGCTACTTCTTCTACTCA-3#, 5#-CTATTTCTTCAT-GAGGAGAA-3#; IspG (AY081261), 5#-ATGGCGACTGGAGTATTGCC-3#,5#-CTACTCATCAGCCACGGGCG-3#; ASN1 (L29083), 5#-AACTCCGAT-

AGCGGCTC-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 cDNAwas 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 63 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-oldArabidopsis leaveswere lysed and separated by centrifugation into

soluble (stroma) and insoluble (membranes) fractions. The ECL1 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 mg/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 mg/mL

kanamycin, 50 mg/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 mg/mL kanamycin, 50 mg/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.

Received December 21, 2004; revised February 26, 2005; accepted February 27,

2005; published April 29, 2005.





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