JXB Advance Access originally published online on July 30, 2004
Journal of Experimental Botany 2004 55(405):1989-1996; doi:10.1093/jxb/erh218
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RESEARCH PAPER |
Accumulation of menaquinones with incompletely reduced side chains and loss of 
-tocopherol in rice mutants with alternations in the chlorophyll moiety
1Department of Plant Resources, Graduate School of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2Department of Materials Engineering, Nagaoka National College of Technology, 888 Nishikatakai, Nagaoka, Niigata 940-8532, Japan
3Forestry Research Institute, Oji Paper Company Ltd., 24-9 Nobono-cho, Kameyama, Mie 519-0212, Japan
* To whom correspondence should be addressed. Fax: +81 92 642 2880. E-mail: ykoba{at}agr.kyushu-u.ac.jp
Received 16 February 2004; Accepted 28 May 2004
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Abstract |
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The rice mutants M249 and M134 accumulate chlorophyllides a and b which are esterified with incompletely reduced alcohols such as geranylgeraniol, dihydrogeranylgeraniol, and tetrahydrogeranylgeraniol. Quantities of
-tocopherol, phylloquinone, and menaquinones in leaves of these mutants were determined by high performance liquid chromatography (HPLC) with a fluorescence detector after post-column chemical reduction to convert quinones to fluorescent quinols. Methylnaphthoquinones, varying in the reduction state of the side chain (menaquinones), were detected in leaf segments of the rice mutants on HPLC analyses with both high selectivity and sensitivity to plant quinones. Mutant M249 preferentially accumulated menaquinone, which contains tetrahydrogeranylgeraniol as its side chain. However, mutant M134 exhibited preferential accumulation of menaquinone with a geranylgeraniol side chain. In both mutants, the accumulation patterns of menaquinones with different prenyl side chains were similar to those of chlorophyll with the corresponding prenyl side chains. The content of P700, the photosystem I primary electron donor, in the wild type was greater than that of either mutant, on both a chlorophyll and a fresh weight basis. However, the ratios of total methylnaphthoquinones to P700 were similar in both the wild type and the mutants. Since no comparative large differences in photosynthetic activity exist between the wild type and the mutants, these results suggest that the hydrogenation of the methylnaphthoquinone side chain to phytol is not an essential requirement for it to function as an electron acceptor in photosystem I. On the other hand, -tocopherol was detected in fully developed leaves of the wild type, but not in those of the mutants. Accumulation of menaquinones and the loss of -tocopherol in mutant leaves suggest that the reduction of chlorophyll-geranylgeraniol to phytol and that of geranylgeranyl pyrophosphate to phytyl pyrophosphate are catalysed by the same enzyme.
Key words:
Chlorophyll, geranylgeraniol, menaquinone, phylloquinone, phytol, -tocopherol
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Introduction |
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On the basis of polyprenyl side chain structure, methylnaphthoquinones (MNQ) can be divided into two main types, phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone, vitamin K1: MNQ-P) and menaquinones (2-methyl-all trans multiprenyl-1,4-naphthoquinone, vitamin K2: MNQ-prenyl) (Fig. 1). Phylloquinone, which is unique to cyanobacteria and higher plants, contains phytol as a side chain and is associated with the reaction centre of photosystem I (PSI) (Schoeder and Lockau, 1986
; Takahashi and Katoh, 1985). The final steps of MNQ-P synthesis consist of the prenylation of 1,4-dihydroxy-2-naphthoate, which is synthesized from shikimate via chorismate and o-succinylbenzoate, and a methylation step by S-adenosylmethionine (Kaiping et al., 1984; Johnson et al., 2000).
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The side chain of chloroplastic MNQ-prenyl is phytol, which is a C-20 prenyl alcohol that is formed by the hydrogenation of geranylgeraniol (GG) (Bartley and Scolnik, 1995
; Suzuki et al., 1997). Geranylgeraniol is distributed as a pyrophosphate ester in the envelope and thylakoid membranes of chloroplasts (Block et al., 1980; Soll and Schultz, 1981). The hydrogenation of geranylgeranyl pyrophosphate (GGPP) in the envelope is distinct from that in the thylakoid membranes. In the envelope, GGPP is reduced in a stepwise manner to phytyl pyrophosphate (PPP) via pyrophosphate esters of dihydrogeranylgeraniol (DHGG) and tetrahydrogeranylgeraniol (THGG). PPP, which is the reduction product, is then used as the substrate of polyprenyltransferase. Polyprenyltransferase subsequently catalyses the addition of a prenyl group to the quinol ring to yield -tocopherol (vitamin E, -Toc) and MNQ-P (the structure of quinones is shown in Fig. 1) (Gaudillère et al., 1984; Joyard et al., 1991; Keller et al., 1998; Soll et al., 1985). In the enzymatic prenylation of the aromatic precursor of either -Toc or MNQ-P, GGPP cannot substitute for PPP (Schultz et al., 1981). This is in contrast to Chl synthetase, which uses GGPP and PPP in the etioplast system (Oster et al., 1997; Rüdiger et al., 1980; Schoefs et al., 2000). Therefore, the last steps in the biosynthesis pathway of MNQ-P in higher plants have been hypothesized to be the hydrogenation of GGPP to PPP and the addition of the phythyl side chain to the aromatic precursor in the envelope; and the methylation and oxidation of quinol to quinone in the thylakoid membranes. However, in contrast to the findings reported by Soll et al. (1983), Kaiping et al. (1984) showed that polyprenyltransferase can use both GGPP and PPP to form prenyl-naphthoquinol in vitro. Furthermore, naphthoquinol with the GG side chain can be readily methylated as MNQ-GG in the thylakoid membranes (Schultz et al., 1981; Kaiping et al., 1984).
On the other hand, in the model systems of Synechocystis, rice, and tobacco, which accumulate Chls with an incompletely reduced side chain, the biosynthetic pathways and physiological functions of the Chl moiety have been determined (Graßes et al., 2001; Havaux et al., 2003; Shibata et al., 2004; Suzuki et al., 1997). In chlorophyll synthesis, GGPP is esterified to chlorophyllide (Chlide) by the enzyme Chl synthetase in the thylakoid membranes and the product Chl-GG is reduced via Chl-DHGG and Chl-THGG to Chl-P by a hydrogenating enzyme (Block et al., 1980; Soll and Schultz, 1981; Benz and Rüdiger, 1981). Analyses of the chloroplastic reductase substrate specificity suggested that the reduction of GGPP to PPP in the envelope and that of Chl-GG to Chl-P in the thylakoid membranes are catalysed by different enzymes (Soll et al., 1983). However, transgenic tobacco plants, in which the activity of GGPP reductase was suppressed by an antisense method, showed a decrease in -Toc content and the accumulation of Chl-GG (Graßes et al., 2001; Havaux et al., 2003; Tanaka et al., 1999). These observations suggested that GGPP is reduced by the hydrogenating enzyme for the Chl moiety, in contrast to the findings reported by Soll et al. (1983). In transgenic tobacco plants, it has been hypothesized that, if prenylation of the MNQ-P precursor 1,4-dihydroxy-2-naphthoate is not specific to PPP, then both MNQ-GG and Chl-GG will be present in leaves. However, transgenic plants have not been examined for MNQs-prenyl.
In order to elucidate the biosynthetic pathway and physiological functions of MNQ-prenyl side chains, the mutants M249 and M134, which were characterized by their ability to accumulate Chlide a and Chlide b esterified with incompletely reduced alcohols, were isolated (Shibata et al., 2004). The amounts of MNQs-prenyl in the leaves of rice mutants M249 and M134 were measured by a highly selective and sensitive method for plant quinones.
In the present study, menaquinones with variations in the reduction state of their side chains accumulated in the leaves of rice mutants M249 and M134. These results suggest that geranylgeranyl pyrophosphate is reduced in a stepwise manner to phytol by the enzyme which catalyses the reduction of the Chl moiety. Furthermore, it is also hypothesized that hydrogenation of the side chain in MNQ-P is not essential to acquire the activity of photosystem I.
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Materials and methods |
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As previously described, Oryza sativa L. cv. Taichung mutants were selected on the basis of their abnormal accumulation patterns of Chlides a and b esterified with incompletely reduced alcohols, such as GG, DHGG, and THGG in leaves (Shibata et al., 2004
). Seeds of wild-type (WT) and mutant plants were sterilized with benomyl solution, and sown in soil. Rice plants were grown for 25 d in a growth chamber at 25 °C with supplemental light containing a photosynthetic photon flux density of 150 µmol photon m–2 s–1. In order to extract Chl and quinones from leaves, rice leaf segments were ground in absolute acetone, and the homogenate was centrifuged. The supernatant was used to measure Chls and quinones. Quantification of Chl intermediates was performed as previously described by Shibata et al. (2004).
For the preparation of C-20 prenyl alcohols GG, DHGG, THGG, and P, Chl intermediates were purified by DEAE Sepharose CL-6B column chromatography (Omata and Murata, 1980) and hydrolysed with 20% (w/v) KOH in ethanol for 30 min at room temperature (Shioi and Sasa, 1983). The liberated side chain alcohols were then dissolved in ethanol for HPLC analysis.
A reverse-phase column (4.6 mm i.d.x25 cm, Zorbax ODS, Shimadzu, Japan) was used for all HPLC analyses. Under normal circumstances, measurement of MNQ-P is carried out by HPLC with a spectrophotometric detector. However, this method lacks both selectivity and sensitivity and measurements of quinones require concentration of the extract and removal of substances that strongly absorb ultraviolet light. Utilization of fluorescence detection methods after HPLC separation has the advantage of the specific detection of MNQs-prenyl. However, this is dependent upon the condition that interfering compounds such as fluorescence quenchers are not co-eluted with the fluorescent compounds. As a result, HPLC was used with fluorescence detection after post-column chemical reduction for the analyses of MNQ-prenyl in mutant leaves without pretreatment of the crude extracts (Hiraike et al., 1988). Naphthoquinone derivatives extracted from leaves were separated by the column at 35 °C with a mixture of an equivalent volume of methanol and ethanol as the mobile phase at a flow rate of 1.0 ml min–1. The quinones were detected with a fluorescence detector (RF-530, Shimadzu, Japan) by monitoring the fluorescence intensity at 430 nm of quinols excited at 320 nm after the chemical reduction of the quinones. Post-column reduction was performed by 0.045% (w/v) ethanolic sodium borohydride at a flow rate of 0.5 ml min–1 in a reaction coil (0.5 mm i.d. x 200 cm) kept at 35 °C connected on-line to the chromatographic reverse-phase column. The calibration curves constructed by plotting the peak height against the amount of authentic MNQ-P or MNQ-GG were linear over the range tested from 2.5 to 1200 pmol. The amounts of MNQs with prenyl side chains were calculated from the peak height by comparison with standard curves obtained for authentic MNQ-P and MNQ-GG under the same running conditions. These calculations were based upon the assumption that fluorescence yield is not altered by the differences in the side chain (Dunphy and Brodie, 1971).
Prenyl alcohols were injected onto the column and eluted at 35 °C with 100% methanol at a flow rate of 1.0 ml min–1, and absorption at 210 nm was monitored with a spectrophotometric detector (SPD-6A, Shimadzu, Japan).
-Tocopherol was eluted at 35 °C with 100% methanol as the mobile phase at a flow rate of 1.2 ml min–1 from the column, and the fluorescence intensity at 335 nm of -Toc excited at 288 nm in the effluent was measured with the fluorescence detector.
The capacity factor (k') was calculated from the equation k'=(tr–to)/(to–ta), which is a simplified version of k'=(tr–ta)–(to–ta)/(to–ta). The abbreviation tr signifies the retention time of a retained compound. The elution time (to) of an unretained component was regarded as equivalent to the elution time of 0.01% NaNO3 solution in the given system (Wells and Clark, 1981). ta stand for the time for passage of the compound in the reaction coil. For this study, each measurement was replicated at least three times.
Absorption spectra of quinones were measured in the eluent with a photodiode array detector (SPD-M10A, Shimadzu, Japan), under the given HPLC conditions without sodium borohydride. A second differential curve was calculated from the obtained spectrum by using the Shimadzu Class-10 program; which serves as the system control program of the HPLC photodiode array and tools developed for peak determination on a personal computer.
With slight modifications, thylakoid membranes were isolated from rice leaves and P700 was determined spectrophotometrically from ferricyanide-oxidized minus ascorbate-reduced difference absorption spectra according to the previously reported method of Terashima et al. (1994). The P700 assay mixture consisted of thylakoid membranes (30 µM Chls), 400 mM sucrose, 10 mM NaCl, 2 mM MgCl2, 20 µM phenazin methosulphate and 0.5 % (w/v) Triton X-100 in 50 mM TRIS-HCl (pH 7.5). In the assay mixture, the concentrations of ferricyanide and ascorbate were 0.36 mM and 1.6 mM, respectively. The differential absorption coefficient of P700 was assumed to be 63 000 M–1 cm–1 at 700 nm.
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Results |
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HPLC analysis of methylnaphthoquinones accumulating in rice leaves
Figure 2 shows HPLC separation profiles of Chls a and MNQs-prenyl extracted from the leaves of WT, M249, and M134. The upper (A, C, and E) and lower traces (B, D, and F) in Fig. 2 are chromatograms obtained by measuring fluorescence at 430 nm excited at 320 nm with and without, respectively, reduction by sodium borohydride. Plant quinones can be selectively measured with a fluorescence detector by reducing quinones to quinols, because quinols have measurerable fluorescence. As peaks 5–8 appeared in the profiles when reductant was applied to the HPLC assay system, the compounds corresponding to these peaks were presumed to be quinones. The main quinones in higher plants are MNQ-P,
-Toc, ubiquinone, and plastoquinone A-45 (Lichtenthaler et al., 1981). Quinones other than MNQ-P and their quinols (reduced forms) were not detectable under the HPLC conditions used to assay MNQs-prenyl. This was most likely due to the observation that absorption and fluorescence spectra of -Toc and plastoquinone differ markedly from those of MNQs-prenyl. Degradation of quinone or quinol species by reductant in the coil was not observed under the conditions used. Therefore, the authors are confident that this HPLC method is useful for the detection of MNQ derivatives in higher plant leaves. A low level of fluorescence of Chl a at 430 nm, excited at 320 nm in a mixture of ethanol and methanol, was observed. However, the fluorescence intensity of Chl a was less than 0.2% of that of MNQ-P on a molar basis.
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In WT, peak 4 was observed by fluorescence detection without using reductant and only peak 8 was detected by using reductant as shown by comparing traces A and B in Fig. 2. When similar analyses were performed for mutants M134 and M249 seedlings, quinone species accumulating in mutant leaves were distinct from those in normal leaves. In M249, three peaks (1, 2, and 3) were observed by normal fluorescence detection (Fig. 2D); while three small peaks (5, 6, and 8) and a large peak (7) were detected by using reductant in the HPLC assay system (Fig. 2C). As shown in Fig. 2E, the separation pattern of extracts from M134 leaves was markedly different from the profiles obtained from WT and M249 samples (traces A and C in Fig. 2). Three main peaks (5, 7, and 8) and a minor peak (6) appeared in the extract from M134 leaves after chemical reduction (Fig. 2E). Retention times of peaks 5 and 8 were in agreement with those of authentic MNQ-GG and MNQ-P, respectively (Table 1). As measured by monitoring fluorescence at 650 nm of Chl a excited at 440 nm, retention times of peaks 1–4 were consistent with those of purified Chl intermediates with incompletely reduced C-20 alcohol side chains. Compared with purified Chl a-P in the eluent, the absorption spectrum of Chls in peaks 1–4 had the same maxima at 432 nm. From these data, peaks 1, 2, 3, and 4 were identified as Chl a -GG, Chl a-DHGG, Chl a-THGG, and Chl a-P, respectively. Under these tested HPLC conditions, little Chl b was detected.
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Absorption spectra of methylnaphthoquinones extracted from leaves
The absorption spectrum of each peak was measured with a photodiode array detector in the HPLC and a second differential curve was calculated from the obtained spectrum. Figure 3 shows the absorption spectra of quinones extracted from leaves of WT and M249 and their analysis with the HPLC system. Absorption spectra of authentic MNQ-P and MNQ-GG in the same system showed maxima at 249, 266, and 272 nm with a high wavelength peak at 332 nm. In agreement with absorption of authentic MNQ-P and MNQ-GG in the eluent, the spectra of quinones in peak 7 (Fig. 2C) and peak 8 (Fig. 2A) showed the same maxima at 249, 266, and 272 nm. As the absorption band at around 330 nm was very small compared with other UV-bands, the wavelength of the maximum in the broad absorption peak between 300 and 350 nm was not determined. The second differential curve of the spectra of the quinones in peaks 7 and 8 showed maxima at 247, 259, 268, and 282 nm (inset in Fig. 3), as did authentic MNQ-P and MNQ-GG (data not shown). It was concluded that the quinone in peak 8 was MNQ-P and that the quinone in peak 7 was MNQ with a prenyl side chain other than P. This conclusion was based on the absence of effects of hydrogenation and length of the side chain on the spectral properties of MNQ-prenyl (Dunphy and Brodie, 1971
). Attempts to measure the absorption spectrum of the quinone in peaks 5 and 6 were unsuccessful. This failure was primarily due to the fact that the UV-absorbance of MNQ-prenyl was hidden by that of Chl intermediates and also because peak 6 was very small.
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Determination of the methylnaphthoquinone side chain
To determine the side chain attached to MNQ in each peak, the capacity factor (k') was calculated from the retention time obtained from Fig. 2 (Table 1). Under the HPLC analysis conditions, the reduction time (ta) of quinones in the coil was 16 s. Figure 4 shows plots of the logarithm of k' calculated from the retention times of Chls, quinones, and diterpene alcohols versus the number of double bonds in the C-20 prenyl side chain. As shown by lines B and C in Fig. 4, the plots from Chls and alcohols produced a straight line with a negative slope, and thereby permitted the estimation of the number of double bonds in the side chains of unknown prenyl C-20 compounds. Assuming that the number of double bonds in the side chains of compounds in peaks 5, 6, 7, and 8 were 4 (MNQ-GG), 3 (MNQ-DHGG), 2 (MNQ-THGG), and 1(MNQ-P), respectively, a linear relationship between the number of double bonds and log k' for each quinone was obtained (line A in Fig. 4). Methylnaphthoquinones with side chains of different length or a modified prenyl chain, such as with addition of a hydroxyl group, did not yield a linear plot (data not shown). From a comparison with the HPLC parameters of authentic MNQ-GG and MNQ-P, the MNQ-prenyl side chains of the compounds in peaks 5–8 were presumed to be diterpene derivatives (C-20 isoprenoids). These results indicated that peaks 5–7 were MNQs with incompletely reduced diterpene side chains, such as GG, DHGG, and THGG, respectively. This identification was taken as evidence to suggest that M249 and M134 are mutants that accumulate MNQ-P intermediates with incompletely reduced side chains.
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In agreement with previous results, which showed that MNQ-P is present only as an oxidized form (quinone type) (Lichtenthaler et al., 1981
), the reduced forms of MNQs-prenyl were not observed in all extracts from WT and the mutants.
Distribution of methylnaphthoquinone and chlorophyll derivatives in rice leaves
Table 2 compares the contents of MNQ and intermediates of Chls a+b in the leaf segments of WT and the mutants based on the analysis described above. M134 leaves preferentially accumulated Chl-GG, while M249 accumulated all Chl intermediates presumed to be produced in the three-step reductions of the Chl alcohol side chain from GG to P. The chlorophyll-phytol content in the mutant leaves was greater than that reported by Shibata et al. (2004) due to some variability in composition between Chl intermediates depending on leaf age and growth conditions of seedlings (data not shown).
The total amounts of MNQs-prenyl, based on leaf fresh weight, decreased in the order WT>M249>M134, although the content of MNQ-P in M249 leaves was less than that in M134 (Table 2). Mutant M249 preferentially accumulated MNQ-THGG, which accounted for 74% of its total MNQs-prenyl. M134 mainly contained three MNQs-prenyl: MNQ-GG, MNQ-THGG, and MNQ-P (the structure is shown in Fig. 1). In the leaves of each mutant, the hydrogenation state of the side chain of the predominant MNQ-prenyl was similar to that of Chl intermediates. However, the relative contents of Chl intermediates were somewhat different from those of MNQs-prenyl. The proportion of more-reduced side chains attached to MNQ was higher than those attached to Chlide. Lastly, there were some variations in the reduction state of the side chain of MNQs-prenyl and Chl. These variations were found to be dependent upon growth conditions and leaf age (data not shown).
Quantification of P700 in thylakoid membranes isolated from leaf segments
As shown in Table 2, the ratio of total Chls to P700 increased in the order WT<M249<M134. Only 12.1% of the total amount of MNQs-prenyl in M249 comprised MNQ-P; whereas, the Chl/P700 ratio in M249 was 1.06-fold that in WT. These results suggest that MNQs with incompletely reduced prenyl side chain moieties can function instead of MNQ-P in the A1 site of PSI. By contrast, the P700 content, based on leaf fresh weight, which was calculated from the total amount of Chls, was approximately 1.6 times higher in WT than in mutants due to the lower Chl content in the mutants. The ratios of the sum of MNQs-prenyl to P700 were 1.89, 1.88, and 1.93 in WT, M249, and M134, respectively (Table 2).
-Tocopherol contents in rice leaves
To determine whether hydrogenation of GGPP to PPP is carried out in the envelope of the rice mutants, -Toc extracted from leaves were measured by HPLC with normal fluorescence detection. Figure 5 shows HPLC separation profiles of -Toc in WT, M249, and M134. -Tocopherol was clearly detected in the extract of WT leaves, but not detected in leaves of either mutant. The detection limit was 0.2 pmol of -Toc under the HPLC conditions used.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Rice mutants M249 and M134 accumulate Chl intermediates with incompletely reduced alcohol side chains due to inhibition of the enzyme that catalyses reduction of Chl-GG to Chl-P in thylakoid membranes (Shibata et al., 2004
). Methylnaphthoquinones with side chains such as GG, DHGG, and THGG were preferentially accumulated in rice mutant leaves (Figs 1, 2). Although menaquinones are widespread in bacteria, they are less common in photosynthetic cyanobacteria and higher plants.
With regard to the enzymatic prenylation of naphthoquinol, Schultz et al. (1981) showed that GGPP could not substitute for PPP as the substrate, by contrast with Chl synthetase which is capable of using GGPP and PPP. However, the rice mutants accumulated MNQs with incompletely reduced side chains (Fig. 2). Measurements of the MNQ-P intermediates accumulating in mutant leaves (Fig. 2; Table 2) suggested that biosynthesis of MNQ-P via menaquinones is capable of taking place in higher plants. In other words, as in the case of Chl synthesis (Oster et al., 1997), GGPP can be used as the substrate for prenylation. However, based only on the profiles of the MNQ-prenyl pool in mutant leaves, it cannot be concluded that MNQ-GG was formed by prenylation of GG to the quinone ring and subsequent hydrogenation of the GG side chain to MNQ-P via MNQ-DHGG and MNQ-THGG. Similarly, it was not possible to determine if MNQs-prenyl with varying degrees of reduction arose through prenylation of incompletely reduced side chains to quinone rings. The prenyltransferase has a relative specificity of 10:1 for PPP:GGPP, and substrate specificity of the methyltransferase is a 3–4-fold preference for naphthoquinol-P over -GG in broken chloroplasts or in the thylakoid membranes (Schultz et al., 1981; Kaiping et al., 1984). Considering the substrate specificity for prenylation and methylation, it is likely that prenylation of P directly to the naphthoquinol ring is the main pathway in chloroplasts (Soll et al., 1983).
As shown in Fig. 2, MNQs-prenyl had side chains of various hydrogenation states, while the total amounts of MNQs-prenyl per P700 in WT, M249, and M134 were similar. Two molecules of MNQ-P, which is the electron acceptor A1, are bound to the reaction centre of PSI (Golbeck, 1987; Takahashi and Katoh, 1985). It has been shown that electron transfer in PSI was not affected by replacing MNQ-P with menaquinone or plastoquinone in A1 (Iwaki and Itoh, 1989; Biggins, 1990; Wade et al., 2000). The P700 assay on the basis of chemical redox does not directly prove a functional PSI in mutants. However, despite the low content (12.1%) of MNQ-P in the total prenyl-MNQ pool in leaves of M249, oxygen evolution was observed in leaves of the mutants as well as WT (Shibata et al., 2004) (Table 2). The most likely explanation for these results is that the side chain of MNQ-prenyl does not play a critical role in the function of A1.
Biggins (1990) suggested that the 3-phytyl side chain of MNQ-P is necessary for the hydrophobic interaction of the quinone with the acceptor site in the core of PSI to promote electron transfer from 
to Fx. The k' value, which is related to the hydrophobicity of compounds with side chains, increased with the degree of hydrogenation of the side chain (lines A, B, and C in Fig. 5). Therefore, when these results are considered collectively with the characteristics of the mutants, it is expected that the MNQ-P molecule is integrated into the A1 quinone binding site and is stabilized by the increase in hydrophobicity depending on the reduction of MNQ-GG to MNQ-P without elongation of the side chain. As there are no large differences in photosynthetic activity between WT and the mutants (Shibata et al., 2004), these results suggest that reduction of the side chain to the phytyl moiety is not essential for assembly of PSI and photosynthesis. Graßes (2001) and Havaux (2003) showed that photosynthetic electron transport was normal in antisense tobacco plants accumulating Chl-GG, due to suppression of GGPP reductase, but MNQ species were not measured.
It was reported by Soll et al. (1983) that the hydrogenation of Chl-GG to Chl-P in thylakoid membranes, and that of GGPP to PPP in the envelope, are catalysed by different enzymes. Furthermore, only PPP which is obtained by hydrogenation of GGPP in the envelope, is used as a substrate of the -Toc side chain. Therefore, it has been thought that inhibition of the hydrogenating enzyme for the Chl moiety does not affect the synthesis of -Toc. However, -TQH was not detected in leaves of either rice mutant, in contrast to WT. These results are consistent with the previous reports that there was a deficiency of -Toc in transgenic tobacco plants accumulating Chl-GG (Tanaka et al., 1999; Graßes et al., 2001; Havaux et al., 2003).
From measurements of MNQs-prenyl, P700, and -Toc in rice mutant leaves and the available data, it was concluded that the hydrogenating enzyme of the Chl moiety can catalyse the stepwise reduction of GG to P. Furthermore, it was concluded that hydrogenation of the side chain in MNQ-P is not essential to achieve the activity of photosystem I.
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Acknowledgements |
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We wish to express our thanks to Professor A Yoshimura (Department of Applied Genetic and Pest Management, Kyushu University) for supplying the rice seeds.
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Footnotes |
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Abbreviations: Chl, chlorophyll; Chlide, chlorophyllide; DHGG, dihydrogeranylgeraniol; GG, geranylgeraniol; GGPP, geranylgeranyl pyrophosphate; HPLC, high performance liquid chromatography; MNQ, methylnaphthoquinone; MNQ-P, phylloquinone; MNQ-prenyl, menaquinone with unspecified prenyl side chain; P, phytol; PPP, phytyl pyrophosphate; PSI, photosystem I; THGG, tetrahydrogeranylgeraniol; -Toc; -tocopherol.
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References |
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