Plant Physiol. (1999) 120: 695-704

Reduced Activity of Geranylgeranyl Reductase Leads to Loss of Chlorophyll and Tocopherol and to Partially Geranylgeranylated Chlorophyll in Transgenic Tobacco Plants Expressing Antisense RNA for Geranylgeranyl Reductase¹

Ryouichi Tanaka², Ulrike Oster, Elisabeth Kruse, Wolfhart Rüdiger, and Bernhard Grimm^*

Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, 06466 Gatersleben, Germany (R.T., E.K., B.G.); and Botanisches Institut der Ludwig Maximilian Universität München, Menzinger Strasse 67, 80638 München, Germany (U.O., W.R.)

	ABSTRACT

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The enzyme geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate. We identified a tobacco (Nicotiana tabacum) cDNA sequence encoding a 52-kD precursor protein homologous to the Arabidopsis and bacterial CHL P. The effects of deficient CHL P activity on chlorophyll (Chl) and tocopherol contents were studied in transgenic plants expressing antisense CHL P RNA. Transformants with gradually reduced Chl P expression showed a delayed growth rate and a pale or variegated phenotype. Transformants grown in high (500 µmol m² s¹; HL) and low (70 µmol photon m² s¹; LL) light displayed a similar degree of reduced tocopherol content during leaf development, although growth of wild-type plants in HL conditions led to up to a 2-fold increase in tocopherol content. The total Chl content was more rapidly reduced during HL than LL conditions. Up to 58% of the Chl content was esterified with geranylgeraniol instead of phytol under LL conditions. Our results indicate that CHL P provides phytol for both tocopherol and Chl synthesis. The transformants are a valuable model with which to investigate the adaptation of plants with modified tocopherol levels against deleterious environmental conditions.

	INTRODUCTION

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The main constituents of the photosynthetic apparatus are Chls, carotenoids, the plastid-encoded apoproteins of the core complex of the reaction centers, and the nuclear-encoded light-harvesting Chl-binding proteins of the antenna complexes. Control of pigment metabolism and expression of pigment-binding proteins ensure a synchronous synthesis of all components in stoichiometric amounts to prevent pigment or protein degradation or photooxidative deterioration.

Chl consists of two moieties, chlorophyllide and phytol, which are formed from the precursor molecules 5-aminolevulinate and isopentenyl diphosphate, respectively, in the two different pathways of tetrapyrrole and isoprenoid biosynthesis. Most of the previous investigations of Chl biosynthesis have emphasized the tetrapyrrolic pathway, which is entirely located in plastids and converts Glu to Chl. Most of the genes involved in tetrapyrrole biosynthesis have been characterized previously (Chadwick and Ackrill, 1994; von Wettstein et al., 1995; Porra, 1997; Rüdiger, 1997; Grimm, 1998).

The branched isoprenoid pathway is rather complex and comprises enzymatic steps in at least two compartments. The cytosolic isoprenoid-synthesizing pathway proceeds from acetyl-CoA via mevalonate in the plant cytoplasm, leading, for example, to sterol compounds. Incorporation studies of labeled early precursors indicated a mevalonate-independent pathway in plastids for phytol biosynthesis, for which 1-deoxy-xylulose-5-P is an intermediate (Rohmer et al., 1993; Lichtenthaler et al., 1997). The exact localization of the pathways for carotenoids and other end products was hampered to a certain extent because isopentenyl diphosphate, which is the common intermediate of both pathways, is transferred through the plastid envelope (Kreuz and Kleinig, 1984; Gray, 1987).

Four molecules of isopentenyl diphosphate are subsequently joined to form the C₂₀-intermediate GGPP, which is then allocated to the synthesis of various end products such as carotenoids, quinones, Chl, or tocopherol. The hydrogenation of GGPP is catalyzed by a CHL P (EC 1.3.1.-). The enzyme Chl synthase links tetrapyrrole and isoprenoid metabolism by esterifying chlorophyllide with GGPP or PhyPP. In etiolated plants Chl synthase esterifies chlorophyllide preferentially with GGPP to form Chl a_GG. The recombinant Chl synthase that is encoded in the G4 gene of Arabidopsis (Gaubier et al., 1995) and overexpressed in Escherichia coli also gives preference to GGPP relative to PhyPP (Oster and Rüdiger, 1997). The NADPH-dependent hydrogenation to the phytol chain of Chl is observed after prenylation of chlorophyllide (Schoch et al., 1977; Benz et al., 1980).

Conversely, Chl synthase assayed from green plants favors PhyPP for Chl a synthesis rather than GGPP (Soll et al., 1983; Rüdiger, 1987). PhyPP is the preferential substrate of two overexpressed proteins, the Chl synthase derived from the chlG gene of Synechocystis and the bacteriochlorophyll synthase encoded by the Rhodobacter bchG gene (Oster et al., 1997).

PhyPP is also an obligatory precursor for the synthesis of tocopherol (Soll and Schultz, 1981), and is directed into the tocopherol-synthesizing pathway by condensation with homogentisate derived from the shikimate pathway. Several methylations and a cyclization step of the quinol intermediate leads to -tocopherol, the major form of the vitamin E fraction (Soll, 1987). Tocopherol prevents the Chl-photosensitized oxidation of thylakoid components, especially when plants are subjected to environmental stress, mainly by quenching activated singlet oxygen or by scavenging oxygen radicals (Fryer, 1992).

A bchP gene has been previously detected as part of the 46-kb photosynthetic gene cluster of Rhodobacter capsulatus (Marrs, 1981; Zsebo and Hearst, 1984) encoding almost all constituents required for photosynthesis. Insertional mutagenesis of the bchP gene resulted in a mutant that produced bacteriochlorophyll esterified with geranylgeraniol instead of phytol (Bollivar et al., 1994). The bchP gene encodes CHL P. Electron transfer and energy transfer from the light-harvesting complex to the reaction center were apparently not affected in the R. capsulatus mutant. Nevertheless, the growth rate under photosynthetic conditions was severely reduced in the mutant (Bollivar et al., 1994). The authors suggested a reduced stability of the pigment-protein complexes, if bacteriochlorophyll is esterified with geranylgeraniol. Other reasons for a reduced growth rate could not be ruled out. Nevertheless, all normal functions were restored when the homologous chlP gene from Synechocystis sp. PCC 6803 was expressed in the Rhodobacter bchP-deficient mutant (Addlesee et al., 1996).

We isolated the tobacco Chl P sequence encoding CHL P and examined its metabolic function as well as its expression in transgenic tobacco plants. CHL P is located at the branch point toward Chl and tocopherol. Our aim was to improve the understanding of the molecular and physiological effects of reduced synthesis of PhyPP on the controlled distribution of substrate for Chl and tocopherol synthesis under different light conditions.

	MATERIALS AND METHODS

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Plant Growth and Harvest

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Isolation and Analysis of a cDNA Clone Encoding CHL P Protein

Construction of a Chl P Antisense Gene and Plant Transformation

RNA Analysis

Antiserum Preparation and Western Analysis

Plant material (100 mg) was ground under liquid nitrogen, suspended in 1 mL of solubilization buffer (56 mM Na₂CO₃, 56 mM DTT, 2% SDS, 12% Suc, and 2 mM EDTA), and denatured for 15 min at 70°C. The soluble protein fraction was quantified and 10-µg protein aliquots were analyzed by western blot with the anti-CHL P antiserum using an immunoblotting kit (ECL, Amersham).

Analysis of Chl

The hexane phase was then repeatedly washed with water until the aqueous phase reached a pH of 5.5. The last traces of water were removed from the hexane phase by freezing at 20°C. Hexane was then removed by evaporation and the precipitate was dissolved in 500 µL of acetone. Twenty microliters of the 1:10 diluted samples was applied for HPLC analysis (model 480, Gynkothek, Ramsey, NJ). Pigments were separated on a column (4 × 250 mm) filled with RP18 (Gromsil 120, Grom Analytic, Herrenberg, Germany) at 1.2 mL/min with the following gradient consisting of 60% acetone (solvent A) and 100% acetone (solvent B): 75% A/25% B for 2 min, followed by 45% A/55% B for 2 min, 30% A/70% B for 11 min, and 100% B for 8 min. Pigments were detected by a UV-visible light detector (model SP5V, Shimadzu, Columbia, MD) at 410 nm and by a fluorescence detector (model RF551, Shimadzu) at 665 nm_em and 425 nm_ex. The peak areas indicated the ratios of Chl a_GG to Chl a_Phy and Chl b_GG to Chl b_Phy.

Analysis of Tocopherol

In the second method, tocopherol was extracted from frozen leaf powder with acetone containing 10 µM KOH and separated on a HPLC system equipped with a C₁₈ column (3.9 × 150 mm, Nova-Pak, Waters) with a gradient of solvent A (30% methanol, and 10% 0.1 M ammonium acetate, pH 5.2) and solvent B (100% methanol) as follows: a linear gradient from 6% A/94% B at 0 min to 1% A/99% B at 10 min until 23 min with the same ratio of solutions A and B. Standards for , /, and  tocopherol and for , /, and  tocotrienol were purchased from Merck (Darmstadt, Germany) and used to quantify and qualify the tocopherol forms eluted by our HPLC program.

Analysis of Carotenoids

	RESULTS

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Tobacco Chl P cDNA Sequence Encoding CHL P and the Expression of Recombinant Protein

The coding region of a truncated CHL P peptide (amino acid residues 50-464) was fused in frame behind the initiation codon into an E. coli expression vector. The beginning of the open reading frame codes for the Met-Gly-Arg-Asn-Leu of the recombinant CHL P. The recombinant protein was insoluble in aqueous solution. The His-tagged protein was dissolved in 8 M urea and was purified by metal chelate affinity chromatography as recommended by manufacturer's instructions. The purified protein of an approximate molecular mass of 47 kD was injected into rabbits for immunization.

Phenotypical Differences between Transgenic Plants Expressing Antisense-Oriented Chl P Genes and Control Plants

The transformants were generally characterized by a growth rate slower than or similar to that of control plants and a gradually reduced Chl content compared with the wild type. Most of the transgenic lines displayed a uniform low pigmentation, and some of them had yellow areas along the leaf veins or different variegation patterns (Fig. 1). The green pigmentation was generally more reduced in older than in younger leaves of the same transformant. The transformants grown in the greenhouse or in the growth chamber under controlled conditions did not show any necrotic leaf lesions that could be generated by accumulating photosensitizing tetrapyrrole intermediates.

View larger version (39K): [in this window] [in a new window]   Figure 1. Primary transformant 6 with Chl P antisense genes (PL24-6) and a wild-type tobacco plant (SNN). Plants were grown for 8 weeks under greenhouse conditions in an average light intensity of 300 µmol m2 s1.

Metabolic Effects of Reduced Expression of Chl P in Transgenic Plants

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The pale-green leaves of some transgenic plants correlated with the reduced total Chl content. The severely affected lines 6, 47, and 10 accumulated only 32%, 37%, and 40%, respectively, of the wild-type Chl content. The transformants also contained a lower tocopherol content. Lines 6, 47, and 10 also showed the strongest deficiency of tocopherol (14%, 18%, and 19% of the control values, respectively). As indicated, other transgenic lines displayed only minor decreases in Chl and tocopherol contents. Tocotrienol could be an expected product if GGPP were also used for the synthesis of tocopherol. The HPLC elution program allowed the separation of tocopherol and tocotrienol derivatives, but tocotrienol was not detected in extracts of the transformants.

The esterification of Chl with various alcohols was analyzed by HPLC. To avoid allomerization and oxidation, we removed the central Mg²⁺ and analyzed the corresponding pheophytins. A typical chromatogram is shown in Figure 2. In the control samples, pheophytins a and b (Phe _Phy and Phe b_Phy) were detectable as the only Chl derivatives. In the samples of the transformants, three new peaks were identified by co-chromatography with authentic samples: the new compound Phe a_Phy and Phe  and Phe b esterified with geranylgeraniol (Phe a_GG and Phe b_GG). The identity of the pigments was confirmed by the absorption and the fluorescence emission spectra of the single peaks and by comparison with the authentic compounds. We could not detect in any of the transformants the pigments containing intermediate alcohols between geranylgeraniol and phytol. This observation of the steady-state content in transgenic plants differs from the results of the in vitro assays with recombinant Arabidopsis CHL P (Keller et al., 1998). The small peaks between those of the identified pigments do not indicate Chl derivatives, but they exhibited only diffuse fluorescence and absorption spectra (Fig. 2).

View larger version (15K): [in this window] [in a new window]   Figure 2. HPLC chromatogram of pheophytins derived from Chls of tobacco leaves. A, Wild-type tobacco; B, tobacco transformed with the Chl P gene in antisense orientation. Labeled peaks are: Phe aPhy (1); Phe aPhy (1a); Phe bPhy (1a); Phe aGG (3); and Phe bGG (4).

The percentages of Chl a_GG, Chl b_GG, Chl a_Phy, and Chl b_Phy were determined by quantification of the peak areas. As expected, the wild-type plants contained only phytylated Chl. Whereas line 21, which had wild-type-like characteristics, contained only 4% Chl_GG, the strongly reduced amounts of Chl of lines 10 and 6 consisted of 56% and 53% Chl_GG, respectively (Table I). If the phytylated portion of Chl was related to the reduced amount of total Chl in the transformants, the values for Chl_Phy and -tocopherol were about equal (Table I), indicating that antisense inhibition of Chl P expression affects the pathways leading to Chl_Phy and to -tocopherol to the same extent.

These initial data of six representative transformants reflect the variation of inhibitory effects by Chl P antisense gene expression in transgenic tobacco plants. The percentage of Chl_GG is apparently a suitable indicator for the extent of CHL P inhibition and was used to substantiate the primary examinations of the antisense inhibition upon different physiological conditions. We grew plants at a constant temperature of 22°C in light intensities of 70 and 500 µM m² s¹ and designated the conditions as LL and HL growth, respectively. All plants exhibited the distinct properties of either LL- and HL-exposed plants apart from their characteristic transgenic phenotype. The LL-grown plants displayed smaller and paler leaves as well as extended internodia. HL-grown plants looked more vigorous and robust and contained more spacious leaves. To test a possible developmental regulation, we analyzed leaves 3 to 10 (counting from the top).

Wild-type leaves contained exclusively Chl_Phy. In contrast, Chl_GG was detected in all transformants under conditions of irradiation and leaf development (Table II). In leaves 1 to 3, Chl_GG was not detectable in any transformant. The percentage of Chl_GG increased generally from leaves 5 to 7 (Table II) and did not increase further in leaves 8 to 13 (data not shown). Therefore, the data indicate an increasing effect of the antisense inhibition with leaf development. Furthermore, the percentage of Chl_GG was generally higher in transgenic plants grown under LL conditions than in the same plants cultivated under HL conditions. Under HL conditions only the three transformants, 6, 10, and 47, showed significant accumulation of Chl_GG in the analyzed leaves (Table II).

No significant difference was found between the portion of Chl a_GG and Chl b_GG in transformants grown under LL conditions. In HL-cultivated transgenic plants 6, 10, and 47, the degree of Chl a_GG was higher than that of Chl b_GG (Table II). As expected, the total Chl a/b ratio was higher in HL-grown wild-type and transgenic plants than in LL-grown plants. In LL-grown plants the Chl a/b ratio did not differ between the geranylgeranylated and the phytylated Chl molecules. Under HL conditions transformants contained a lower degree of Chl b_GG than Chl a_GG, resulting in a higher Chl a/b ratio for the geranylgeranylated molecules (5.2 in leaf 5 of transformant 47, 6.3 in leaf 5 of transformant 6, and of 7.5 in leaf 7 of transformant 10).

Chl P Expression in CHL P-Deficient Transgenic Plants under Two Different Light Intensities

View larger version (62K): [in this window] [in a new window]   Figure 3. Expression of CHL P in transgenic (6, 10, and 20) and wild-type tobacco plants (SNN) was determined by northern and western analysis of HL- and LL-grown plants. Total RNA was isolated from leaves 3, 5, and 7 (counted from the top of each plant). Ten micrograms of RNA was loaded per lane, separated on a 1% formaldehyde-agarose gel, and hybridized to a tobacco Chl P cDNA probe. A cDNA probe for 18S rRNA was subsequently hybridized to the RNA on the same filter. Equal amounts of protein extracted from leaf 3, 5, and 7 were loaded on a SDS-polyacrylamide gel. After transfer to a nitrocellulose filter, immunodetection was performed with antiserum raised against recombinant CHL P.

Chl, Tocopherol, and Carotenoid Contents Are Developmentally Controlled in Transgenic Plants under LL and HL Conditions

View larger version (45K): [in this window] [in a new window]   Figure 4. Contents of total Chl (C and D) and -tocopherol (A and B) in extracts of leaf 3, 5, 7, and 10 from LL-grown (A and C) and HL-grown (B and D) transgenic tobacco plants with reduced CHL P expression (lines 6, 10, and 20) and control plants (SNN). White bars, Leaf 3; right-hatched bars, leaf 5; left-hatched bars, leaf 7; and cross-hatched bars, leaf 10. fw, Fresh weight.

Tocopherol content was analyzed in HL- and LL-grown control and transgenic plants (Fig. 4). Evaluation of the tocopherol content in premature leaves of control plant and of lines 20, 10, and 6 revealed that under HL conditions the plants contained 110%, 83%, 139%, and 47%, respectively, more tocopherol than under LL growth conditions. In wild-type plants the tocopherol content increased maximally up to leaf 7 under both light intensities, remained constant during plant development, and decreased progressively during senescence (data not shown). Tocopherol content was progressively reduced from the young to the older leaves in lines 6 and 10 and only slightly lower in line 20 after a similar profile during leaf development, compared with wild-type plants. The degree of lowered tocopherol levels was different in each transformant, but the relative decrease in tocopherol contents was very similar in each respective line under conditions of either LL or HL growth (Fig. 4). In leaf 10 of lines 6 and 10, tocopherol yielded approximately 20% of the wild-type contents under both growth conditions and in line 20 the yield was approximately 75%.

Comparison of the decline in Chl and tocopherol in the transgenic relative to the wild-type plants during leaf development revealed that the relative tocopherol contents were diminished to a similar extent under both light intensities and that the relative amount of total Chl was more intensively reduced with age in HL-grown than in LL-grown plants of the same transgenic lines.

The total carotenoid contents during leaf development of LL-and HL-grown transformants paralleled approximately the total Chl content. Accumulation of the carotenoid species neoxanthin, violaxanthin, lutein, and -carotene is depicted in Table III. The amounts of zeaxanthin and antheraxanthin could not be determined by our HPLC program because Chl a_GG was eluted from the HPLC column simultaneously with zeaxanthin and Chl b_GG with antheraxanthin. All carotenoid species accumulated to lower amounts in the transgenic plants grown under HL or LL conditions than in control plants. However, the reduction of lutein and -carotene content was more pronounced in the transgenic plants relative to wild-type plants than the reduction of violaxanthin and neoxanthin. Comparing the contents of lutein and -carotene during developmental growth of the transgenic lines, it is noticeable that both carotenoids were more diminished under HL than under LL growth conditions.

	DISCUSSION

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The plastidal metabolite GGPP is an intermediate in several biosynthetic pathways. It can be channeled into the Chl pathway by reduction to PhyPP and final esterification with chlorophyllide or vice versa by the initial prenylation of chlorophyllide and a subsequent reduction of Chl_GG (Fig. 5). GGPP can also be directed into tocopherol synthesis when PhyPP is condensed with homogentisate. CHL P catalyzes the reduction of GGPP and Chl_GG in vitro (Keller et al., 1998). It is not known if the enzyme accepts both substrates with the same specificity. Moreover, it remains to be seen if the same enzyme serves simultaneously the Chl- and the tocopherol-synthesizing pathways in planta.

View larger version (17K): [in this window] [in a new window]   Figure 5. Scheme of the branched pathway starting from GGPP to -tocopherol, -tocopherol, or ChlPhy. CHL P, Chl synthase, and an isoprenyl transferase are indicated. CHL P uses GGPP and ChlGG as substrates and directs PhyPP to the tocopherol- and the Chl-synthesizing pathway.

We identified the tobacco Chl P cDNA sequence and introduced a binary vector harboring an antisense Chl P gene into tobacco to reduce specifically the enzyme activity of CHL P. This transgenic approach not only addresses the question of whether CHL P functions for two pathways but also enables a prediction on the control mechanism that distributes the enzymatic product of CHL P toward Chl or tocopherol synthesis.

The antisense inhibition of the Chl P expression led to an increasing proportion of Chl_GG in the total amount of Chl and to lower tocopherol contents during leaf development of the analyzed transformants (Table II). The deficiency of tocopherol was found synchronously with the increasing amount of Chl_GG (Table I). Therefore, the involvement of CHL P in the formation of Chl and tocopherol can be described as two equivalent functions. Our results obtained with the Chl P antisense plants are in agreement with the assumption of hydrogenation of GGPP to PhyPP and of Chl_GG to Chl_Phy and with the second function of CHL P protein in contributing to the formation of tocopherol. The recombinant Arabidopsis CHL P protein expressed in E. coli was shown to be active in both the reduction of GGPP and of Chl_GG (Keller et al., 1998). The R. capsulatus bchP insertion mutant fails in the hydrogenation step from geranylgeraniol to phytol, the esterifying alcohol of bacteriochlorophyll (Bollivar et al., 1994).

Under increased light intensities, wild-type plants and the analyzed transformants accumulated more Chl (between approximately 35% in leaves of wild-type plants and 12% in those of line 6) and up to 2 times more tocopherol (Fig. 4). Since the wild-type contents of CHL P seemed to be similar under LL and HL conditions in leaves of the same age, it is suggested that CHL P expression does not normally limit the supply of precursor for Chl and tocopherol during plant development. However, as was apparent from the analysis of the transgenic lines, significant reduction of the amount of CHL P by antisense RNA synthesis affects the levels of both biomolecules in a light and developmentally dependent manner. It is remarkable that transformants with diminished amounts of CHL P not only contained reduced contents of phytylated Chl but also contained less total Chl than wild-type plants. It is assumed either that Chl_GG is not associated as stably as Chl_Phy in the photosynthetic pigment-binding proteins, resulting in a faster Chl breakdown (analogous to the assumption of labile bacteriochlorophyll_GG-protein complexes; Bollivar et al., 1994), or that the reduced synthesis of isoprenoid and Chl precursors is indirectly caused by the antisense inhibition of CHL P expression.

The percentage of phytylated Chl was generally higher in HL transformants than in the same LL-grown transgenic lines, which could be explained by several factors. First, if Chl synthase activity were higher under HL than under LL conditions, more PhyPP but also more accumulating GGPP could be channeled into the Chl-synthesizing pathway. Chl_GG competes with GGPP for the residual hydrogenation activity of CHL P, leading to more Chl_Phy. Second, an increasing pool of NADPH or other reducing biomolecules due to the stimulated photosynthesis in HL-exposed leaves could activate CHL P, which should lead to more Chl_Phy and -tocopherol. However, it seems more likely that the low Chl_GG levels in HL-grown transgenic plants result from photooxidation of Chl_GG so that Chl_Phy remains preferentially. It is unknown whether HL conditions cause destabilization of pigments, especially of Chl_GG in the Chl P antisense plants.

Chl a and b were phytylated to the same extent in most transformants grown under LL conditions. Some transformants exposed to HL conditions showed more Chl a_GG than Chl b_GG, which is indicative for a preferential supply of Chl for the reaction center core complex under increasing light intensities. This would mean that Chl a exhibits a faster turnover than Chl b in HL-exposed transformants, most likely in the reaction center of the photosystems and to lesser extent in the antenna complexes.

Approximately 2 times more tocopherol accumulated upon increased light intensity in wild-type and transgenic plants compared with those exposed to lower light intensities. It has been shown previously that HL exposure modulates the tocopherol content in photosynthetic membranes (Lichtenthaler, 1979). The antioxidant function of tocopherol is expected to scavenge photogenerated singlet oxygen or other organic radicals in photosynthetic membranes. Exposure to enhanced light intensities increases the risks of photodynamic damage. An increased requirement of tocopherol under HL conditions is most likely adjusted by stimulation of its synthesis in response to light intensities. The analysis of the transgenic lines with reduced CHL P expression did not provide evidence that HL exposure would lead to preferential channelling of PhyPP under limiting synthesis of this precursor into the tocopherol-synthesizing pathway. However, the consequences of insufficient levels of tocopherol became apparent in the transgenic plants with reduced CHL P content. The transformants grew slower and showed a bleached phenotype.

Lower CHL P activity could result in accumulation of GGPP, which can also be directed into carotenoid synthesis. However, the analyzed carotenoid levels were lower in the transformants than in the wild-type plants and almost paralleled the reduction in Chl content. The assembly of Chl_GG instead of Chl_Phy with the pigment-binding proteins does not significantly modify the composition of carotenoids in the pigment-protein complexes. Our observation reflects the synchronized need for both pigment fractions and the pigment-binding proteins to stabilize the photosynthetic complexes and is consistent with observations of the parallel loss of pigments and pigment-binding proteins in mutants with deficiencies in Chl or carotenoid synthesis (Plumley and Schmidt, 1995).

In conclusion, we demonstrate here, for the first time to our knowledge, that deficiency of CHL P simultaneously affects two pathways, leading to a decline in tocopherol and phytylated Chl contents and in the level of total Chl molecules. Lack of tocopherol could destabilize the thylakoid membrane and negatively influence the photosynthetic machinery. Analysis of the photoprotective functions of tocopherol and other antioxidants under various environmental conditions and for photosynthesis in the Chl P antisense plants is currently being performed. Transgenic plants with reduced tocopherol contents seem to be an appropriate model with which to investigate how the transformants prevent the deleterious photo effects and if and how they compensate for the tocopherol deficit.

	FOOTNOTES

   Received February 5, 1999; accepted April 14, 1999.

	ABBREVIATIONS

Abbreviations: Chl, chlorophyll. CHL P, geranylgeranyl reductase. GGPP, geranylgeranyl diphosphate. HL, high light. LL, low light. PhyPP, phytyl diphosphate.

	ACKNOWLEDGMENTS

We thank P. Linow, E. Fraust, and I. Blos for excellent technical assistance.

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