RNAi-Mediated Tocopherol Deficiency Impairs Photoassimilate Export in Transgenic Potato Plants

RNAi-Mediated Tocopherol Deficiency ImpairsPhotoassimilate Export in TransgenicPotato Plants1

Daniel Hofius*, Mohammad-Reza Hajirezaei, Michael Geiger2, Henning Tschiersch, Michael Melzer,and Uwe Sonnewald

Institut fur Pflanzengenetik and Kulturpflanzenforschung (IPK), D–06466 Gatersleben, Germany(D.H., M.-R.H., H.T., M.M., U.S.); and Sungene GmbH & Co. KGaA, D–06466 Gatersleben, Germany (M.G.)

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and thephotosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have beenproposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherolcyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacksa phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in thisstudy, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a proteinwith high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescentprotein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption ofTC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence oftocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to bea consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosyntheticgene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting acarbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity.This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreasedtocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from sourceleaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherolreduction.

The Suc export defective1 (sxd1) mutant from maize(Zea mays) has long been thought to represent the onlyknown mutant showing altered plasmodesmata (PD)structure and function during leaf development. Thisassumption was based on a typical photosynthateexport-deficient phenotype characterized by an over-all growth reduction and source leaf-specific accumu-lation of anthocyanins and starch. In addition, minorveins of maturating sxd1 leaf blades exhibited ultra-structural alterations and callose occlusion of a specificclass of PD between bundle sheath (BS) and vascularparenchyma (VP) cells (Russin et al., 1996; Botha et al.,2000). This suggested an inhibition of symplasticcontinuity at the BS-VP boundary, leading to a blockof Suc transport into the phloem. Indeed, symplastictransport of the fluorescent dye Lucifer Yellow intominor veins was specifically blocked in anthocyanin-

accumulating source regions of sxd1 leaf blades (Bothaet al., 2000). Therefore, it was tempting to speculatethat the respective mutation would reside in a genethat is essential for PD function. However, afteridentification of the sxd1 locus, SXD1 turned out tobe a novel chloroplast-targeted protein of unknownfunction. Thus, a model was proposed that SXD1 isinvolved in a chloroplast-to-nucleus signaling path-way in maize BS cells essential for PD formationduring sink-source transition (Provencher et al., 2001).

Recently, evidence for the function of SXD1 has beenprovided by genetic analysis of the Arabidopsis vte1mutant, which was discovered during a mutant screenfor altered tocopherol (vitamin E) content and compo-sition (Porfirova et al., 2002). In general, tocopherolsare amphipathic molecules that are composed ofa polar chromanol head and a hydrophobic isoprenoid(prenyl) tail. Therefore, substrates for tocopherolbiosynthesis are drawn from two different metabolicpathways, the shikimate pathway and the plastid-localized nonmevalonate pathway. The first commit-ted step in tocopherol biosynthesis is the condensationof homogentisate and phytyl pyrophosphate by ho-mogentisate phytyl transferase to yield 2-methyl-6-phytyl-1,4-hydroquinone (Collakova and DellaPenna,2001; Savidge et al., 2002). Subsequent ring methyl-ation and ring cyclation reactions lead to the formation

1 This work was funded by a grant from the Deutsche For-schungsgemeinschaft (DFG; SO 300/6–1).

2 Present address: BASF Plant Science GmbH, D–67056 Ludwig-shafen, Germany.

* Corresponding author; e-mail [email protected]; fax0049–39482–5515.

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.104.043927.

1256 Plant Physiology, July 2004, Vol. 135, pp. 1256–1268, www.plantphysiol.org � 2004 American Society of Plant Biologists

of the four major tocopherol derivates (a-, b-, g-, andd-tocopherol) that differ only in number and positionof methyl substituents on the aromatic ring. The vte1mutant lacked all four derivatives of tocopheroland was devoid of tocopherol cyclase (TC) activity(Porfirova et al., 2002). Genetic mapping of vte1combined with a genomics-based approach identifiedVTE1 as a gene encoding TC. Surprisingly, the se-quence of VTE1 shared a high degree of similarity toSXD1 and, based on Arabidopsis genome and maizeexpressed sequence tag (EST) database analysis, it wassuggested that VTE1 and SXD1 represent single-copyorthologs, both encoding an enzyme with TC activity(Porfirova et al., 2002). Lately, the functional equiva-lency of SXD1 and VTE1 could be verified unequivo-cally by different approaches (Sattler et al., 2003). First,sxd1 mutant leaves showed tocopherol deficiency andaccumulated 2,3-dimethyl-5-phytyl-1,4-hydroquinone(DMPQ), a substrate of TC; second, recombinant SXD1protein exhibited TC activity in vitro; and third,expression of maize SXD1 in the Synechocystis sp.strain PCC 6803 protein slr1737 knockout mutantcomplemented the lack of tocopherol cyclase activityand restored vitamin E synthesis (Sattler et al., 2003).

Despite the molecular and biochemical similaritiesof VTE1 and SXD1 in vte1 and sxd1 mutant leaves, theSuc export phenotype was absent in vte1 Arabidopsisplants (Sattler et al., 2003). Thus, unlike the sxd1mutant, vte1 did not accumulate carbohydrates andanthocyanins and did not exhibit stunted growth.In addition, chlorophyll content and photosyntheticefficiency of vte1 plants were very similar to wild typeunder optimal growth conditions (Porfirova et al.,2002). However, during photooxidative stress at highlight conditions, chlorophyll content and photosyn-thetic quantum yield decreased slightly as com-pared to wild type (Porfirova et al., 2002). These dataappeared to be in good agreement with the well-defined in vitro ability of tocopherols to scavenge andquench reactive oxygen species and lipid peroxyradicals by physical and chemical means (for review,see Fryer, 1992; Munne-Bosch and Alegre, 2002).

The mechanistic link between tocopherol deficiencyand the formation of aberrant PD at the BS-VP in-terface leading to the Suc transport defect in the sxd1maize mutant is completely unknown. Moreover, thereasons for phenotypic differences between Arabidop-sis and maize remain elusive. Sattler et al. (2003)postulated that additional biological activities of to-copherols, which are not related to their antioxidantfunction, might be involved in the different responsesof Arabidopsis and maize to tocopherol deficiency.There is accumulating evidence from studies in mam-malian systems that specific tocopherols are able tomodulate signal transduction pathways or act assignal molecules themselves (for review, see Azziet al., 2002; Rimbach et al., 2002). Although directevidence for analogous nonantioxidant roles of tocoph-erols in plants is lacking, it was speculated thattocopherol deficiency in sxd1 interfered with signal

transduction events that are required specifically forthe formation of BS-VP PD (Sattler et al., 2003). Thistocopherol-dependent signaling pathway might bedifferent or absent in Arabidopsis due to anatomicaland physiological differences between C3 and C4and/or between monocot and dicot species.

To address the principal question of whether thesecondary effects of tocopherol deficiency on PDfunction and photoassimilate transport are restrictedto the sxd1 mutant from maize and thus could berelated to special features of C4 plants, comparativeexperiments on different plant systems are required.Therefore, we have chosen potato (Solanum tuberosumL. var. Solara) as an alternative model plant. Potatorepresents a C3 crop plant and is one of the best-characterized plants with respect to sink-sourcerelations and carbohydrate metabolism (Stitt andSonnewald, 1995; Herbers and Sonnewald, 1998). Inthis study, the SXD1 ortholog from potato was isolatedand functionally analyzed in vitro as well as in plantausing an RNAi-silencing approach. We could demon-strate that the reduction of tocopherol cyclase enzymeactivity in transgenic potato lines resulted in tocoph-erol deficiency and caused a characteristic photoas-similate export-deficient phenotype, most likely due tovascular-specific callose deposition in source leaves.These data provide evidence that the impact of to-copherol deficiency on PD function and carbohydratemetabolism is similar in both monocot and dicotspecies and thus cannot be assigned to specific ana-tomical or biochemical features of C4 metabolism.Furthermore, the absence of comparable phenotypicchanges in the Arabidopsis vte1 mutant suggestssignificant differences in the C3 plant family in re-sponse to tocopherol deficiency.

RESULTS

Isolation of SXD1 cDNA from Potato and EnzymaticActivity of Recombinant Protein

To identify a potato ortholog of maize SXD1(ZmSXD1) and Arabidopsis VTE1 (AtSXD1) proteins,EST databases were searched for homologous se-quences using tBLASTn (BLAST; Altschul et al., 1990).A partial EST sequence derived from mature potatotubers (accession no. BF460070) was amplified byreverse transcription (RT)-PCR from potato leaf mate-rial. After obtaining 5# and 3# sequences by RACEtechniques, the full-length cDNA, designated StSXD1(accession no. AY536918) was isolated and fully se-quenced. StSXD1 encoded a 56.2-kD protein with 501amino acids that shared approximately 62% aminoacid identity with the TCs from maize and Arabidop-sis, respectively, and also showed substantial similar-ity to predicted TCs from different photosyntheticbacteria, including the Synechocystis sp. protein en-coded by open reading frame slr1727 (34% amino acididentity), that was previously shown to be a functionalTC (Sattler et al., 2003).

Silencing of Tocopherol Cyclase in Potato

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To provide evidence that the potato SXD1 orthologcodes for a functional protein with TC activity,the StSXD1 cDNA was heterologously expressed inEscherichia coli and used for enzyme assays. SinceStSXD1 contained in accordance with ZmSXD1 andAtSXD1/VTE1 (Provencher et al., 2001) an apparentN-terminal transit peptide (TP) for chloroplast target-ing, the precursor protein (StSXD1), as well the pre-dicted mature part lacking the N-terminal 89 aminoacid (StSXD1DTP), was analyzed. As demonstrated inFigure 1A, recombinant StSXD1 and StSXD1DTP pro-teins showed high TC activity with the substratesDMPQ and 2,3-dimethyl-5-geranlygeranyl-1,4-hydro-quinone (DMGQ), leading to the synthesis of g-to-copherol and g-tocotrienol, respectively. This clearlydemonstrated that StSXD1 is a functional TC whoseactivity is not influenced by the N-terminal targetingsignal in vitro.

StSXD1 Is Targeted into Chloroplasts

To analyze the functionality of the predictedN-terminal TP in planta, intracellular localization offull-length StSXD1, as well as the N-terminal portion(89 amino acid) fused to green fluorescent protein(GFP; Chalfie et al., 1994), respectively, was deter-mined after transient expression in epidermal cells oftobacco (Nicotiana tabacum) source leaves. As depictedin Figure 1, B to J, StSXD1-GFP appeared to be targetedinto chloroplasts since it colocalized in the mergedpicture with the red fluorescence of chlorophyll. Tran-sient expression of the StSXD1 TP-GFP fusion resultedin a similar chloroplastic targeting, whereas expres-sion of GFP alone revealed only cytosolic localization.These data indicate that StSXD1 contains a functionalTP and is targeted into chloroplasts.

RNAi-Mediated Silencing of StSXD1 Resultsin Tocopherol Deficiency in Potato Plants

To functionally analyze the in planta role of StSXD1,transgenic potato plants constitutively expressing anintron-spliced hairpin RNA (RNAi) construct targetedat StSXD1 were generated (pBin-StSXD1-RNAi; Fig.2A). Twenty-eight kanamycin-resistant transformantswere screened for reduction in StSXD1 transcript levelby northern-blot analysis (data not shown). Fourtransgenic plants showing strong silencing, desig-nated StSXD1-RNAi-14, -21, -22, and -27, respectively,and, in addition, StSXD1-RNAi-18 with similar tran-script levels as nontransformed wild-type plants, wereselected and vegetatively multiplied in tissue culturefor biochemical analysis. Five weeks after transfer intothe greenhouse, TC activity and tocopherol contentswere determined in source leaves of transgenic lines.In correlation with decreased StSXD1 transcript levels,TC activity was suppressed in lines 14, 21, 22, and 27compared to wild type, respectively, leading to dra-matically reduced vitamin E levels as indicated bydetermination of the 2 predominant tocopherol deriv-

atives in leaves, a- and g-tocopherol (Fig. 2, B and C).Among the selected lines, StSXD1-RNAi-22 showedthe strongest repression of TC activity, resulting ina tocopherol-deficient phenotype (0.7% of a-tocoph-erol wild-type levels), comparable to the Arabidopsisvte1 null mutant. StSXD1-RNAi-27 and -21 were alsostrongly affected (2.1% and 4.1% of a-tocopherol wild-

Figure 1. TC activity of recombinant StSXD1 protein in E. coli andintracellular targeting of StSXD1 in leaves. A, The premature (columns3 and 6) and mature part (columns 2 and 5) of StSXD1 cDNA wasexpressed in E. coli cells and used for TC assay with DMGQ andDMPQas substrates. The products, g-tocotrienol and g-tocopherol, werequantified by fluorescence HPLC. The values represent the mean ofthree independent experiments and SD. E. coli cells transformed withan empty vector pQE11 served as control (columns 1 and 4) andaccumulated less than 2 ng g-tocotrienol or 1 ng g-tocopherol/mg ofprotein. B–J, GFP fusion proteins of full-length StSXD1 (StSXD1:GFP)and N-terminal TP (StSXD1TP:GFP) as well as GFP protein alone weretransientlyexpressed in tobaccoepidermalcellsbymicroprojectilebom-bardment and analyzed by confocal microscopy after 20 h. Green colorindicates GFP fluorescence and red color reveals chlorophyll (Chl)fluorescence. B, Intracellular localization of StSXD1:GFP in terminaltrichome cell. C, Chl fluorescence indicates chloroplast distribution. D,Merged image. E, Intracellular localization of StSXD1TP:GFP instomata cell. F, Chl fluorescence of chloroplasts. G, Merged image.H, Intracellular localization of free GFP in stomata cell. I, Chlfluorescence of chloroplasts. J, Merged image. Bars represent 10 mm.

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type levels, respectively), whereas line 14 showed anintermediate phenotype (34.5% of a-tocopherol wild-type levels). In contrast to these significantly silencedlines, StSXD1-RNAi-18 plants exhibited only a minordecrease in TC enzyme activity and slightly lowereda-tocopherol content (80.9% of a-tocopherol wild-type levels) and thus was subsequently included asa weakly affected control line. Apparently in closecorrelation with the inhibition of TC activity and thereduction of tocopherol content, transgenic linesshowed an accumulation of the TC substrate DMPQ(Fig. 2D), which was absent in wild-type plants andthus indicated a specific disruption of TC by silencingof StSXD1. Collectively, these results provide evidencethat the SXD1 ortholog from potato codes for a func-tional TC in planta involved in the biosynthesis oftocopherols.

Tocopherol Deficiency Leads to ImpairedPhotoassimilate Export

To evaluate whether tocopherol deficiency inStSXD1-silenced potato lines resulted in a Suc exportdefect similar to the maize sxd1 mutant phenotype(Russin et al., 1996; Provencher et al., 2001) or did notalter carbohydrate metabolism as observed in Arabi-dopsis vte1 plants (Sattler et al., 2003), steady-statelevels of soluble sugars and starch were determined.To this end, the set of StSXD1-RNAi transgenic linescharacterized for inhibition of TC activity and tocoph-erol reduction before (compare Fig. 2) was analyzed in 3different leaf stages (leaf 11, 8, and 3 from the top) afteran additional 4-week growth period. As indicated inFigure 3A, 59-d-old plants of line StSXD1-RNAi-22,showing the strongest reduction in TC activity andtocopherol content, accumulated massive amounts ofcarbohydrates in lower source leaves (leaf 11) at the endof the dark period. Starch and soluble sugar levelsincreased 9-fold and 4.1-fold, respectively, compared towild-type levels. A considerable accumulation in starchand soluble sugar content was also detectable forStSXD1-RNAi-27 plants (3.7-fold and 1.8-fold, respec-tively), whereas starch levels in lines 21 and 14 wereslightly, but not significantly, enhanced relative to wild-type plants. In upper source leaves (leaf 8), carbohy-drate status remained dramatically increased in line 22,but was not significantly altered in line 27, as well as inlines 21 and 14, respectively, whereas in young de-veloping leaves (leaf 3) overall carbohydrate levels didnot accumulate in any of the transgenic lines. These dataclearly suggested a leaf age-dependent occurrence ofthe phenotype potentially correlating with the sink-source gradient in the plant. To analyze the spatialdistribution of phenotype development in more detail,leaves of different developmental stages from line 22were assessed for starch accumulation by iodine stain-ing at the end of the dark period. As depicted in Figure3B, the starch-excess phenotype appeared to be sourceleaf-specific and restricted to the nonvascular regions ofthe leaf, indicating a defect in photoassimilate export.

Figure 2. RNAi-mediated silencing of StSXD1 leads to suppression of TCactivity and results in tocopherol deficiency as well as DMPQ accumu-lation in transgenic potato plants. A, Schematic structure of the binaryintron-spliced hairpin RNA (RNAi) expression construct used for trans-formation of potato plants. StSXD1 fragments (765 bp, nts 503–1,268 ofStSXD1 cDNA; accession no. AY536918) in sense and antisense orien-tation separated by intron 1 of potatoGA20 oxidase (200 bp) were placedbetween the cauliflower mosaic virus 35S promoter and the ocs termi-nator of the Bin19-derived vector using the indicated restriction sites. B,TC activity. C, Tocopherol contents (a- and g-tocopherol). D, DMPQlevels in source leaves of StSXD1-silenced transgenic lines (StSXD1-RNAi-18, -14, -21, -27, and -22) compared to wild type. TC activity wasdetermined using DMGQ as substrate and tocopherols, as well as theprenyl quinoneprecursorDMPQ,werequantifiedby fluorescenceHPLC.Samples were taken from source leaves 5 weeks after transfer form tissueculture and data represent the means6 SE of 5 individual plants.



To further elucidate whether the observed exportimpairment of source leaves would be limited to carbonor would also affect nitrogen allocation, amino acidcontents andcomposition weredetermined at the end ofthe photoperiod. Remarkably, total amino acid levels inlower source leaves (leaf 11, see above) increased 3.7-fold in StSXD1-RNAi-22 plants and were also enhanced(between 1.5- and 2.1-fold) in the other transgenic lines(Fig. 4). The strong increase in amino acid levels ofStSXD1-RNAi-22 was mainly due to dramatically ele-vated levels of the osmoprotectant Pro, which accumu-lated 24.8-fold compared to wild type (Fig. 4). Gln to Gluratios were also strikingly enhanced in lines 22 and 27,indicating a shift toward the transport forms of aminoacids. Although Asn to Asp ratios were not comparablyaltered (data not shown), the increase in the Gln to Gluratio correlated well with starch accumulation in therespective transgenic lines (compare Fig. 3A), suggest-ing animpaired export capacityof source leaves bothforcarbohydrates and amino acids.

Despite the inhibition of photosynthate transportfrom source leaves, overall stature and growth oftransgenic plants was not substantially changed com-pared to the control (Fig. 5A). However, carbohydrate-accumulating source leaves of line 27, and especiallyline 22, developed phenotypic alterations character-ized by chlorotic interveinal regions, leaf curling,and necrotic spots at the leaf margins (Fig. 5B).Additionally, tuber yield was significantly reduced in

line 22 and slightly lowered in line 27, compared towild-type plants, respectively, which provided cir-cumstantial evidence that leaves of the strongest af-fected lines fail to effectively export photoassimilatesfor storage sink development (Fig. 5C).

Tocopherol Deficiency Causes Enhanced CalloseDeposition in the Vascular Tissue

The assimilate export-deficient phenotype in themaize sxd1 mutant has previously been attributed tocallose occlusion of PD at the BS-VP interface of matu-rating leaf blades, thereby preventing the symplastictransport of Suc into the phloem tissue (Botha et al.,2000; Provencher et al., 2001). Therefore, callose con-centration and distribution were analyzed in the samelower source leaves of tocopherol-deficient potato linesin comparison to wild type as used for photoassimilatedetermination before (leaf 11; compare Figs. 3 and 4).Consistent with the development of the starch-excessphenotype, callose accumulated in source leaves ofStSXD1-silenced plants and reached highest levels inlines 22 and 27 (2.1-fold and 1.6-fold, respectively)relative to wild type. Using a fluorescein isothiocyanate-labeled b-1,3-glucan antibody and confocal laser scan-ning microscopy, the callose localization pattern wasvisualized in leaf sections of the most strongly affectedline, StSXD1-RNAi-22, in comparison to the control. Asshown in Figure 6B, enhanced callose deposition in

Figure 3. Carbohydrate steady-statelevels and spatial distribution ofstarch accumulation in leaves oftocopherol-deficient potato plants.A, Soluble sugar and starch contentsin three different leaf stages (11th,8th, and 3rd leaf from the top) of59-d-old plants at the end of the darkperiod. Two samples were takenfrom each leaf and averaged; valuesare given as means 6 SE of 5 inde-pendent plants, and soluble sugarsrepresent the sum of Glc, Fru, andSuc. B, Qualitative determinationof starch accumulation in leaves ofStSXD1-RNAi-22 and wild type afteran extended dark period (16 h)by iodine (KI) staining. Bars repre-sent 2 cm.

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tocopherol-deficient plants was obvious as fluorescingaggregates decorating the cell wall of phloem-associ-ated cells, whereas comparable vascular cell types ofcontrol plants showed only very little callose-derivedfluorescence. In contrast, nonvascular mesophyll cellsof both StSXD1-silenced plants and wild-type controlsdid not exhibit significant callose labeling. These resultsdemonstrated that, in agreement with the sxd1 maizemutant, enhanced callose synthesis and depositionwere restricted to the vascular tissue in StSXD1-sup-pressed potato plants and, therefore, might similarly bethe reason for the disruption of assimilate export.

The Assimilate Export-Deficient Phenotype IsAccompanied by Changes in Photosynthetic

Capacity and Altered Gene Expression

The absence of tocopherol in the vte1 mutant haspreviously been shown to have no major impact onphotosynthetic performance under optimal growthconditions (Porfirova et al., 2002). On the other hand,

it is well established that carbohydrate accumulationin leaves results in reduced rates of photosynthesis bydirect or indirect feedback inhibition mechanisms(Krapp and Stitt, 1995; Herbers et al., 1997; Paul andPellny, 2003). Therefore, we investigated whethertocopherol deficiency and/or photosynthate accu-mulation affected CO2 assimilation in lines StSXD1-RNAi-21, -27, and -22 compared to wild type.Gas-exchange rates were determined in 3 differentleaves (corresponding to leaf stages 11, 8, and 5 in Fig.3), which were chosen according to the occurrence ofthe starch-excess phenotype in line 22 and includedyounger leaves that did not exhibit starch accumu-lation (Fig. 7A). As depicted in Figure 7B, maximumphotosynthetic rates of StSXD1-RNAi-22 plants weresignificantly reduced by 50.3% in lower and 33.7% inupper source leaves as compared to equivalent leavesof wild-type plants under approximately saturatinglight conditions (1,500 mM photons m22 s21). In con-trast, CO2 assimilation was significantly enhanced by11.7% in young leaves of line 22 in comparison to wild-type leaves, suggesting compensatory effects in devel-oping leaves that lack the assimilate export-deficientphenotype. Apparently consistent with the smallerdegree of carbohydrate accumulation in lines 27 and21, respectively, reduction of CO2 exchange rates inlower and upper source leaves of the respective linesrelative to wild type was less pronounced and not assignificant as observed for line 22. Accordingly, anincrease in photosynthesis in young leaves comparedto the control was not obvious in StSXD1-RNAi-27 andStSXD1-RNAi-21 plants, respectively. In contrast tomaximal photosynthetic rates at saturating light con-ditions, the initial slope of the light response curvesappeared to be less affected in tocopherol-deficientlines relative to wild type. A significant reduction ofCO2 exchange rates at low light intensities was onlydetectable in lower source leaves of phenotypic lines,whereas upper source leaves as well as young leaveswere largely unaltered in this respect. Hence, thesystem of light reactions, including light harvestingand electron transport rate, was only compromised inthe oldest starch-accumulating leaf stage. Taken to-gether, these results indicated that carbohydrate accu-mulation rather than tocopherol deficiency per seinfluenced photosynthetic performance of StSXD1-silenced potato plants.

Previously, it was observed that sugar accumulationin leaves, whether as a result of impaired assimilateexport or of sugar feeding, negatively regulated theexpression of photosynthetic genes and induceddefense-related gene expression (Herbers et al., 1996a,1996b; Rolland et al., 2002). However, the sxd1 maizemutant failed to down-regulate the accumulation of vari-ous photosynthetic proteins (Provencher et al., 2001).Therefore, photosynthetic (rbcS and cab) gene expres-sion was studied by northern blotting in the samelower and upper source leaves of StSXD1-silencedplants as used for carbohydrate analysis before(see Fig. 3). Figure 8 shows that rbcS and cab tran-

Figure 4. Amino acid concentrations in source leaves of StSXD1-silenced potato plants. Leaf samples were taken at the end of the lightperiod from lower source leaves (leaf 11 from the top) of 59-d-oldplants. Total amino acid levels represent the sum of single aminoacid concentrations determined by HPLC. Values are given as means(n 5 4) 6 SE.



script accumulation was reduced in lower sourceleaves compared to wild type. In upper source leaves,suppression of photosynthetic gene expression oc-curred in StSXD1-RNAi-22 plants in correlation withexcess sugar accumulation (see Fig. 3), whereas theother lines showed no effect on rbcS expression or evenexhibited a slight increase of cab transcripts (lines 21and 27). In addition, transcripts of the defense-relatedproteinase inhibitor II (Pin2), previously shown to beup-regulated by sugars and jasmonic acid (JA; Johnsonand Ryan, 1990; Wasternack and Parthier, 1997), werestrongly induced in phenotypic source leaves ofStSXD1-RNAi-22 and -27. Possible changes of JA-related gene expression in response to tocopheroldeficiency were further supported by the slight tran-scriptional up-regulation of the JA biosynthetic geneallene oxide cyclase (AOC) in these lines. Consistentwith the observed Pro accumulation (Fig. 4), an in-crease of transcript levels was also detectable for thekey enzyme in Pro biosynthesis, D1-pyrroline-5-car-boxylate synthase (P5CS), which was demonstrated tobe inducible by Suc and abscisic acid (Hellmann et al.,2000; Abraham et al., 2003). Together, these indicatedcarbohydrate- and potentially stress-induced alter-ation of gene expression in tocopherol-deficient potatoplants.

DISCUSSION

Tocopherols are believed to represent key compo-nents of the antioxidant network in the chloroplast andto play a protective role against toxic free radicalsgenerated during photosynthesis and various abioticstresses (Fryer, 1992; Munne-Bosch and Alegre, 2002).However, most of our understanding of tocopherolfunctions has been derived from nonplant systems,

since tocopherols (vitamin E) are essential dietarycomponents for humans and other mammals. Recentefforts to identify the enzymatic steps involved in thetocopherol biosynthetic pathway and to improvevitamin E contents in crop plants by metabolic engi-neering have provided a number of different mutantand transgenic plants for the analysis of tocopherolfunction in planta (Shintani and DellaPenna, 1998;Collakova and DellaPenna, 2001, 2003; Porfirova et al.,2002; Savidge et al., 2002; Bergmuller et al., 2003;Cheng et al., 2003; Van Eenennaam et al., 2003). In thisrespect, it was discovered that the maize sxd1 mutant,originally isolated based on a block in Suc transportfrom leaves (Russin et al., 1996; Provencher et al.,2001), and the Arabidopsis vte1 mutant were bothdefective in the TC gene, resulting in a complete lackof tocopherols (Porfirova et al., 2002; Sattler et al.,2003). However, the photoassimilate export-defectivephenotype caused by aberrant PD formation andcallose deposition in BS cells of sxd1 maize plantswas absent in the vte1 Arabidopsis mutant, whichsuggested additional nonantioxidant and signaltransduction-related functions of tocopherols in C4plants (Sattler et al., 2003). Here, we show that RNAi-mediated disruption of tocopherol cyclase activity inthe C3 crop plant potato results in a severe tocopherol-deficient phenotype comparable to the orthologousArabidopsis and maize mutants. As a consequence,transgenic potato plants exhibited a characteristic pho-toassimilate export defect as seen in the sxd1 mutant,which appeared to be likewise caused by vascular-specific callose deposition in source leaves. Therefore,the impact of tocopherol reduction on callose accu-mulation, assimilate transport, and carbohydratemetabolism occurs both in C4 and C3 plants. Addi-tionally, the lack of an sxd phenotype in Arabidopsis

Figure 5. Growth response, leaf morphol-ogy, and tuber yield of tocopherol-deficientpotato plants. A, Visible phenotype of trans-genic lines and wild type after 9 weeks inthe greenhouse. Bar represents 50 cm. B,Phenoytpic alterations of lower sourceleaves from StSXD1-RNAi-22 and -27 com-pared to wild type. Arrowheads indicatenecrotic spots at the leaf margins. C, Tuberyield of StSXD1-RNAi and wild-type plantsgrown for 13 weeks in the greenhouse. Datarepresent means 6 SE of 5 plants.

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suggests significant and potentially nonantioxidant-related differences between C3 species in their pleio-tropic response to tocopherol deficiency.

StSXD1 Encodes a Functional Chloroplast-TargetedTC Enzyme

The isolated potato ortholog of the SXD1 gene wascharacterized in vitro and in planta, providing severallines of evidence that StSXD1 is functionally equiva-lent to the recently identified TCs from Arabidopsis(AtVTE1/AtSXD1) and maize (ZmSXD1). First, heter-ologous expression of StSXD1 in E. coli resulted in thesynthesis of a protein with high TC activity as revealedby conversion of DMPQ and DMGQ into g-tocopheroland g-tocotrienol, respectively (Fig. 1A). A compara-ble activity has previously been detected for recombi-nant AtSXD1/AtVTE1 and ZmSXD1 proteins,respectively (Porfirova et al., 2002; Sattler et al.,2003). Second, the N-terminal domain of StSXD1,which shared only a few conserved amino acid resi-dues with other SXD1 plant homologs, targets StSXD1to the chloroplast as revealed by transient expressionof GFP-tagged fusions in tobacco leaves (Fig. 1B). Thischloroplastic localization of StSXD1 is consistent withthe results obtained by chloroplast import assays forZmSXD1 (Provencher et al., 2001), as well as with thereported TC activity and tocopherol biosynthesis inplastids (Soll et al., 1985; Arango and Heise, 1998).Third, silencing of StSXD1 resulted in a loss of TCactivity and caused the accumulation of the TC sub-strate DMPQ as well as tocopherol deficiency in leavesof transgenic potato plants, indicating that StSXD1 isrequired for tocopherol biosynthesis (Fig. 2, A–D).

Severe Tocopherol Deficiency Is Required to Induce

a Blockage of Photoassimilate Export

Efficient silencing of StSXD1 was obtained by ex-pression of an intron-spliced hairpin RNA (RNAi)construct, which led to the generation of three stronglyaffected lines with less than 5% of wild-type a-tocoph-erol level (Fig. 2C). However, the induction of a severeSuc export-deficient phenotype characterized by a sev-eralfold increase of carbohydrate contents and mor-phological alterations in source leaves, as well asa strong reduction in tuber yield, was only detectablein line StSXD1-RNAi-22 (Figs. 3 and 5), which nearlyshowed a complete lack of tocopherols. Slightly highertocopherol contents resulted in a considerably smallerextent of phenotype development in plants of line 27or affected assimilate export only marginally as ob-served for line 21. This indicated a low threshold leveland the requirement of a comparably strong impair-ment of tocopherol biosynthesis as obtained by thesxd1 null mutation to induce the assimilate transportblock. The phenotype of StSXD1-RNAi-22 plants wasreminiscent of transgenic potato plants inhibited inapoplastic phloem loading of Suc by antisense repres-

Figure 6. Callose concentrations and distribution in source leaves oftocopherol-deficient potato plants. A, Callose contents in lower sourceleaves (leaf 11 from the top) of 59-d-old potato plants at the end of thephotoperiod. Data represent the means (n 5 5) 6 SE and are given asb-1,3-glucan pachyman equivalents. B, Immunofluorescence analysisof vascular-specific callose accumulation in StSXD1-RNAi-22 com-pared to wild type. Samples were taken from lower source leaves of59-d-old potato plants and callose was detected in longitudinal sectionusing a monoclonal anti-b-1,3-glucan antibody and Alexa Fluor 488antimouse IgG as fluorescence marker. Callose-derived fluorescencesignals in vein class III-associated cells were detected by confocal laserscanning microscopy and superimposed with bright field images. Barsrepresent 50 mm.



sion of the Suc transporter SUT1 (Riesmeier et al., 1994;Kuhn et al., 1996). However, it is unlikely that carbo-hydrate accumulation in tocopherol-deficient plantsresulted from Suc transporter malfunction, becausethe block in carbon export was accompanied byimpaired amino acid transport as revealed by theaccumulation of total amino acids and severalfoldincrease of Gln to Glu ratios in source leaves (Fig. 4).These effects concur well with the assumption that theblockage of the symplastic transport route into minorveins, as demonstrated previously by dye-couplingmicroinjection studies for sxd1 mutant leaves (Bothaet al., 2000), would likewise affect carbon and nitrogen

allocation. The increase of Pro contents in sourceleaves of phenotypic line StSXD1-RNAi-22, and lesspronounced of line StSXD1-RNAi-27, further indi-cated an enhanced stress response as a consequenceof tocopherol reduction and/or excess carbohydrateaccumulation. Pro has been shown to function asa protective compatible osmolyte and to accumulatein response to osmotic stress conditions stimulated bysalt, cold, drought, and abscisic acid (Hare et al., 1999;Hasegawa et al., 2000), as well as by elevated hexosecontents (Bussis et al., 1997). Consistently, the pathwayfor Pro biosynthesis appeared to be up-regulated asevidenced by increased gene expression of P5CS inphenotypic lines (Fig. 8).

Tocopherol Deficiency Indirectly Affects Photosynthetic

Capacity by Carbohydrate-MediatedFeedback Mechanisms

The assimilate export defect in StSXD1-silencedplants was paralleled by a significant decrease inphotosynthetic capacity (Fig. 7). This effect appearedto be leaf age-dependent and directly linked to thesource leaf-specific carbohydrate accumulation, sinceyoung leaves that lacked a starch-excess phenotypeeither were not affected (StSXD1-RNAi-21, 27) or evenshowed an increase in CO2 exchange rates for StSXD1-RNAi-22. Additionally, CO2 assimilation at lower lightintensities indicated that the light reaction was onlyaffected in lower carbohydrate-accumulating sourceleaves and, thus, was not generally inactivated bytocopherol deficiency. These results provide clearevidence that tocopherol per se is not essential forphotosynthetic performance at optimal growth condi-tions, which concurs well with previous results ob-tained for the Arabidopsis vte1 mutant (Porfirova et al.,2002) and for partially tocopherol-deficient tobaccoplants repressed in the expression of geranylgeranylreductase (Grasses et al., 2001). A decrease in photo-

Figure 7. Photosynthetic capacityin tocopherol-deficient potatolines. A, Starch accumulation inthree different leaf stages (11th,8th, and 5th leaf from the top) ofwild type and StSXD1-RNAi-22plants selected for CO2 gas-ex-change measurements. B, Light re-sponse curves of StSXD1-RNAi-21,-27, and -22 plants in comparisonto wild type. Photosynthetic rateswere determined at 400 mM M

21

CO2 concentration using a LICORLI6400 instrument. Plants were an-alyzed 8 weeks after transfer to thegreenhouse and data representmeans 6 SE of 4 plants.

Figure 8. Gene expression analysis of StSXD1-silenced plants. TotalRNA was isolated from lower and upper source leaves (11th and 8thleaves from the top of 59-d-old StSXD1-RNAi lines and control plants(wild type), and transcript levels were analyzed by northern blotting.Thirty micrograms of RNA were loaded per lane and hybridized withcDNA probes of photosynthetic (rbcS, cab), defense-related (Pin2), aswell as JA (AOC) and Pro (P5CS) biosynthetic genes. Additional probingwith an 18S cDNA served as loading control.

Hofius et al.


synthetic rate has frequently been observed inresponse to soluble sugar and starch accumulationindicative of feedback regulation of photosynthesis(Riesmeier et al., 1994; Kuhn et al., 1996; Herbers et al.,1997; Paul and Pellny, 2003, and references therein).Aside from the possibility of impaired chloroplast func-tion as a consequence of excessive starch accumula-tion, carbohydrate-mediated feedback inhibition ofphotosynthesis has been attributed to hexose-inducedrepression of photosynthetic genes via hexokinase-dependent and -independent signals (Herbers et al.,1996a, 1996b; Rolland et al., 2002). Accordingly, rbcS-and cab-specific transcripts appeared to be down-regulated in source leaves of StSXD1-RNAi-22 plantsthat accumulated large amounts of soluble sugars inaddition to starch (Fig. 3A). Consistently, in the ab-sence of sugar accumulation as evident for uppersource leaves of lines 21 and 27 (compare Fig. 3),photosynthetic transcripts were either not affected(rbcS) or even slightly up-regulated (cab). The occur-rence of carbohydrate-mediated changes of gene ex-pression was further supported by the induction ofPin2 transcripts in sugar-accumulating source leaves,which is in good agreement with the former hypoth-esis that suppression of photosynthetic genes andactivation of defense-related genes might be initiatedby common sugar-sensing mechanisms (Herbers et al.,1996a; Rolland et al., 2002). However, despite a similaraccumulation of soluble sugars, mature leaves of thesxd1 maize mutant failed to alter photosynthetic geneexpression (Provencher et al., 2001) as observed inStSXD1-silenced potato plants. Although the exactreason for this discrepancy is unknown, it might bedue to either different threshold levels of solublesugars for repression of photosynthetic genes in maizeand potato plants or the general differences of CO2assimilation in C4 and C3 plants.

Callose Deposition in the Vascular Tissue Correlateswith Tocopherol Deficiency and the Defect inPhotoassimilate Export

The assimilate export phenotype in source leavesof StSXD1-repressed transgenic lines correlated wellwith the enhanced formation and vascular-specificdeposition of callose (Fig. 6). This observation ap-peared to be in close agreement with previous datafrom sxd1 mutant leaves demonstrating that calloseocclusion of PD at the BS-VP interface preventedsymplastic transport into the phloem (Botha et al.,2000), and thus resulted in the inhibition of photosyn-thate export. Although plasmodesmal permeability ofcallose-accumulating vascular cells in StSXD1-silencedpotato plants was not analyzed in this study, it is likelythat the observed cell wall deposition of callose wentalong with PD closure, leading to the interruption ofsymplastic Suc transport and finally excess assimilateaccumulation.

There is compelling evidence in the literature thatPD represent highly regulated channels in the cell wall

that dynamically respond to external and internal stim-uli by altering their size exclusion limits (Haywoodet al., 2002; Roberts and Oparka, 2003). In this re-spect, the formation of callose deposits at the neckregion of PD leading to reduced size exclusion limitsor PD closure has frequently been described as a spe-cific response of plants to different abiotic and bioticstresses, including wounding, metal toxicity, and mi-crobial infection (Enkerli et al., 1997; Iglesias andMeins, 2000; Sivaguru et al., 2000; Roberts and Oparka,2003). For instance, enhanced callose accumulationwas observed in membranes and PD of pea roots inresponse to aluminum treatment, leading to the block-age of symplastic transport and inhibition of rootelongation (Sivaguru et al., 2000). Interestingly, calloseformation was strongly correlated with aluminum-induced oxidative damage to membrane lipids andchanges in intracellular calcium homeostasis, suggest-ing a mechanistic link between lipid peroxidation andcallose synthesis (Jones et al., 1998; Yamamoto et al.,2001). Because tocopherols are proposed to functioneffectively in the scavenging of lipid peroxy radicalsresponsible for the propagation of polyunsaturatedfatty acid oxidation (Wefers and Sies, 1988; McKersieet al., 1990; Fryer, 1992; Munne-Bosch and Alegre,2002), it is tempting to speculate that tocopherol de-ficiency may indirectly affect callose synthesis byincreasing the extent of lipid peroxidation in thechloroplast membranes. Apparently consistent withthis view, it has recently been postulated that tocoph-erols play a role in intracellular signaling by control-ling accumulation of lipid hydroperoxides, which arederived from lipid peroxidation and used for JAbiosynthesis (Schaller, 2001; Munne-Bosch and Alegre,2002; Munne-Bosch and Falk, 2004). Thus, tocopherollevels may indirectly regulate the amount of JA in theleaves and influence JA-dependent gene expressioninvolved, for instance, in the wound stress response.The induction of JA-responsive Pin2 transcripts, aswell as the slight up-regulation of JA biosynthetic geneAOC in severely tocopherol-deficient potato plants,might support this hypothesis. Interestingly, a callosesynthase gene (GSL5) has recently been isolated fromArabidopsis, which is required for wound- and path-ogen-induced callose formation (Jacobs et al., 2003;Nishimura et al., 2003). Further research is needed todetermine whether callose-synthesizing activities areindeed responsive to lipid peroxidation-derived or JA-dependent signals and whether other hormones (e.g.salicylic acid) involved in stress signaling, and poten-tially in the regulation of callose synthesis (Østergaardet al., 2002), are also affected by tocopherols, assuggested recently (Munne-Bosch and Penuelas, 2003).The absence of lipid peroxidation in vte1 Arabidop-sis plants at optimal growth conditions (Bergmulleret al., 2003) might lead to the tempting hypothesisthat the crop plant potato and the mustard weedArabidopsis differ in their basal stress tolerance andsensitivity of membrane lipids to oxidative damageunder tocopherol deficiency. However, a detailed anal-



ysis of lipid composition and malondialdehyde levelsin StSXD1-silenced potato plants, indicative of the ex-tent of lipid peroxidation, is required to draw any fur-ther conclusions in this respect. Similarly, based on thevarious antioxidant-independent functions of tocoph-erols known from mammalian systems (Brigelius-Flohe and Traber, 1999; Rimbach et al., 2002), it ispossible that tocopherol or the prenyl quinone pre-cursor DMPQ acts as signaling molecules and modu-lators of other stress-unrelated signal transductionpathways in plants, as discussed in detail by Sattleret al. (2003). These functions might differ significantlybetween the C3 species Arabidopsis and potato andthus have to be equally considered for future attemptsto unravel the molecular basis of callose depositionand impaired assimilate export in response to tocoph-erol deficiency.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Solanum tuberosum L. var. Solara (potato) was obtained from Bioplant

(Ebstorf, Germany) and maintained in tissue culture under a 16-h-light/8-h-

dark period (150 mM m22 s21 light, 21�C) at 50% relative humidity on

Murashige and Skoog medium (Sigma, St. Louis) containing 2% (w/v) Suc.

Plants used for biochemical and physiological analysis were multiplied

vegetatively in tissue culture and then grown in soil either under controlled

conditions (70% humidity, 16-h day/8-h night regime, 19�C/15�C, 500 mM m22

s21 light) in a growth chamber or in the greenhouse with supplementary light

(250 mM m22 s21 light).

Cloning of StSXD1 and Heterologous Expressionin Escherichia coli

A partial 765-bp EST clone (accession no. BF460070) homologous to

Arabidopsis VTE1 and maize (Zea mays) SXD1 cDNAs was amplified by

RT-PCR from potato leaf material using M-MLV [H-] reverse transcriptase

(Promega, Madison, WI) and oligo(dT)[30] V primer for reverse transcription as

well as rTaq-Polymerase (TaKaRa Shouzo, Japan) and gene-specific primers

(D184, 5#-GGATCCGGGAGCTCAAATTCTTGGTGCAGATG-3#; D185, 5#-GTCGACAATGTGGTTCCATGATCTTTTGAAG-3#) for PCR amplification.

The resulting PCR fragment was subcloned into pCR2.1 vector (Promega) and

sequenced to verify the identity of the StSXD1 clone. 5# and 3# fragments were

obtained by RACE using the SMART RACE cDNA amplification kit (CLON-

TECH, Palo Alto, CA) and StSXD1-specific primers (StSXD1-5#RACE,

5#-CTCTGTGCGCGGCGATTGAACTCCTGAGG-3#; StSXD1-3#RACE, 5#-GGATTAAGGCGACTTCCGGGATTGAATGAG-3#). Finally, the full-length

StSXD1 cDNA clone was amplified by RT-PCR from potato leaves using

RACE fragment-deduced oligonucleotides (D278, 5#-AGTTGCCGCTCCT-

CAAAAACTCTACAWTCC-3#; D279, 5#-TTCTGCTATACTAGCAATAAGA-

TTTTCCAT-3#), and the entire sequence was determined (accession no.

AY536918).

For heterologous expression in Escherichia coli, the StSXD1 precursor

(nucleotides [nts] 53 to 1,555) as well as the mature part without the apparent

signal peptide (StSXD1DTP, nts 320–1,555) was PCR amplified from the

full-length cDNA clone using the oligonucleotides D292 (5#-GGGATCCC-

CATGGAGAGCTTTTATAGTGTTTCCGC-3#) and D291 (5#-GTCGACTC-

AAAGGCCAGGAGGTTTGAGAAGTGAAG-3#) for StSXD1, and D290

(5#-GGGATCCGGACTCCTCATAGCGGGTATCATTTTG-3#) and D291 for

StSXD1DTP, respectively. The PCR fragments were ligated into the BamHI

and SalI sites of pQE11 and transferred into E. coli M15(pREP4) cells for

protein expression (Qiagen, Hilden, Germany). Recombinant E. coli cells were

harvested 3 to 4 h after induction with 1 mM isopropylthio-b-galactoside,

resuspended in 0.1 M potassium phosphate buffer (pH 8.0), and disrupted by

sonification. After additional washing of the cell suspension (30 min at 4�C)

with potassium phosphate buffer containing 2% (v/v) CHAPS, the insoluble

cell debris was removed by centrifugation and the soluble protein fraction was

used for enzymatic measurements.

Construction of GFP Fusion Proteins andMicroprojectile Bombardment

To obtain GFP fusion constructs of StSXD1 for transient expression in leaf

tissue, the complete coding region of StSXD1 (nts 53–1,555), as well as the

predicted TP (StSXD1TP) including the 5#UTR (nts 1–334) were PCR amplified

from the full-length cDNA clone using oligonucleotides D292 (5#-GGGATC-

CCCATGGAGAGCTTTTATAGTGTTTCCGC-3#) and D296 (5#-GTCGACAA-

GGCCAGGAGGTTTGAGAAGTGAAG-3#) for StSXD1, and D311 (5#-GGA-

TCCAGTTGCCGCTCCTCAAAAACTCTA-3#) and D312 (5#-GTCGACCC-

CGCTATGAGGAGTTCGAAGAGG-3#) for StSXD1TP, respectively. After

subcloning into pCR-blunt, the BamHI/SalI-digested PCR fragments were

inserted into the BamHI/SalI sites of a pFF19-based vector (Timmermans

et al., 1990) containing mGFP5 instead of the GUS gene (pFF19-GFP kindly

provided by A. Wachter, Heidelberg) to yield plasmids pFF19-StSXD1:GFP

or pFF19-StSXD1TP:GFP. Plasmid DNA was coated onto 1-mm gold particles,

and the abaxial epidermis of tobacco (Nicotiana tabacum) source leaves was

bombarded using a Helios Gene Gun following the manufacturer’s proto-

col (Bio-Rad Laboratories, Hercules, CA). Images were obtained with a con-

focal microscope LSM 510 META (Zeiss, Gottingen, Germany). Excitation

light of 488 nm produced by krypton/argon laser and emission filters of 510

to 525 and 645 to 700 nm allowed detection of GFP or chlorophyll-derived red

fluorescence, respectively, and images were superimposed by means

of the Zeiss LSM Version 3.0 software. Transient expression of the empty

pFF19-GFP plasmid served as a cytosolic control.

RNAi Plasmid Construction and Potato Transformation

The aforementioned 765-bp PCR fragment of the StSXD1 gene comprising

nts 503 to 1,268 and flanked by BamHI/SalI restriction sites (underlined in

primers D184 and D185, respectively) was inserted in sense orientation

downstream of the GA20 oxidase intron in the pUC-RNAi vector as described

by Chen et al. (2003). The same fragment was subsequently placed in antisense

orientation into the XhoI/BglII sites of pUC-RNAi already carrying the

StSXD1 sense fragment. Finally, the entire RNAi cassette comprising sense

and antisense fragments of StSXD1 interspersed by the GA20 oxidase intron

was excised from pUC-RNAi using the flanking PstI sites and inserted into the

SbfI site of pBinAR (Hofgen and Willmitzer, 1990) between the cauliflower

mosaic virus 35S promoter and ocs terminator yielding the construct pBin-

StSXD1-RNAi.

Transformation of potato plants by Agrobacterium-mediated gene transfer

using Agrobacterium tumefaciens strain C58C1:pGV2260 was carried out as

described previously (Rocha-Sosa et al., 1989).

RNA Analysis

Extraction of total RNA from leaf material and northern-blot analysis was

performed as described by Chen et al. (2003). Thirty micrograms of RNA per

sample were separated on a 1.5% (w/v) formaldehyde-agarose gel, transferred

to a nitrocellulose membrane (GeneScreen, NEN Life Science Products,

Boston), and hybridized with random-primed [a-32P]-labeled cDNA frag-

ments. Photosynthetic gene-specific (rbcS, cab) cDNAs used for hybridization

were described previously (Herbers et al., 1996a, 1996b). Pin2 (accession no.

AY129402) and AOC (accession no. AJ272026) cDNAs from tomato (Lycoper-

sicon esculentum) were kindly provided by C. Wasternack (Halle/Saale,

Germany). A partial tobacco EST clone showing 97% identity to the tomato

P5CS gene (accession no. U60267) was additionally used as cDNA probe. Gel

blots were hybridized with 18S rRNA for loading control.

TC Enzyme Assay and Quantification of Tocopherol

TC activity was determined in protein extracts of E. coli cells and leaves

using DMPQ and DMGQ as substrates following the method as described by

Porfirova et al. (2002). Tocopherol contents were determined after methanol

extraction by fluorescence HPLC as described before (Porfirova et al., 2002).

The immediate tocopherol precursor DMPQ was determined in the same leaf

Hofius et al.


extracts as used for tocopherol analysis and quantified based on peak area

(mV min21 g21 fresh weight) using an authentic standard.

Carbohydrate and Amino Acid Determination

Soluble sugars and starch levels were determined in leaf samples ex-

tracted with 80% (v/v) ethanol/20 mM HEPES-KOH, pH 7.5 as described

(Sonnewald, 1992). Starch accumulation was visualized by iodine staining

(0.3% [w/v] I2, 0.7% [w/v] KI) after an extended dark period (16 h) and

removal of chlorophyll by 80% (v/v) ethanol at 55�C.

Total amino acids, Pro, Gln, and Glu were analyzed in 25-mg leaf samples

extracted with 80% (v/v) ethanol, 20 mM HEPES-KOH, pH 7.5. Extracts were

evaporated to dryness, redissolved in purest H2O, and separated on a re-

versed-phase HPLC system (Waters Associates, Milford, MA) after derivati-

zation using the AccQ-Tag method as described (Rolletschek et al., 2002).

Amino acids were identified by cochromatography with authentic standards

and quantified by comparison with internal standards.

Callose Determination andImmunofluorescence Analysis

Extraction and measurement of callose from leaf material was done as

described (Kohle et al., 1985). Callose quantification was based on comparison

with the fluorescence of known amounts of the commercial b-1,3-glucan

pachyman (ICN Biomedicals, Irvine, CA), and measurements were performed

using a SpectraMax Gemini XS spectrofluorometer (Molecular Devices,

Ismaning, Germany). Callose contents were expressed as b-1,3-glucan pachy-

man equivalents.

For immunofluorescence detection of callose, potato leaf samples were

fixated, substituted, and embedded as described previously in Bornke et al.

(2002). Semithin sections with a thickness of approximately 1 mm were

mounted on slides and immunolabeling was performed by using antimouse

monoclonal (1,3)-b-D-glucan antibody (Biosupplies, Parkville, Australia) as

primary antibody and Alexa Fluor 488 goat antimouse IgG (Molecular Probes,

Eugene, OR) as fluorescence marker. Images were taken with the confocal

microscope Zeiss LSM 510 META and fluorescence signals were detected after

excitation with a 488-nm krypton/argon laser using an emission filter of 505 to

530 nm.

CO2 Gas-Exchange Measurements

CO2 uptake rates were measured with a portable photosynthesis system

LI-6400 (LI-COR, Lincoln, NE). CO2 concentration of the air entering the leaf

chamber was adjusted to 400 mM M21 and leaf temperature was maintained at

20�C. For determination of light response curves of CO2 gas exchange,

photosynthetic photon flux density was varied between 0 and 2,000 mM

photons m22 s21.

Sequence data from this article have been deposited with the EMBL/

GenBank data libraries under accession number AY536918.

ACKNOWLEDGMENTS

We especially thank Anita Winger and Ulrike Schlereth for excellent

technical assistance, Bernhard Claus for skillful help with confocal laser

scanning microscopy, and Andrea Knospe for plant transformation and tissue

culture work. We are also grateful to Prof. Dr. Claus Wasternack (Martin-

Luther-University, Halle/Saale, Germany) for providing the Pin2 and AOC

cDNA fragments, and to Dr. Andreas Wachter (Heidelberg) for the pFF19-

GFP vector.

Received April 1, 2004; returned for revision April 23, 2004; accepted April 23,

2004.

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