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J. Biol. Chem., Vol. 281, Issue 22, 15385-15393, June 2, 2006

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Pigment Shuffling in Antenna Systems Achieved by Expressing Prokaryotic Chlorophyllide a Oxygenase in Arabidopsis^*

Masumi Hirashima, Soichirou Satoh, Ryouichi Tanaka, and Ayumi Tanaka¹

From the Institute of Low Temperature Science, Hokkaido University, Kita-ku, N19 W8, Sapporo 060-0819 and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Received for publication, March 28, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The first step of photosynthesis is to harvest light energy, a feat that is accomplished by various photosynthetic pigments. Photosynthetic pigments bind to apoproteins and form pigment-protein complexes (1) that enable the efficient energy transfer between pigment molecules and the construction of super complexes of antenna systems. Photosynthetic light-harvesting systems are divided into the categories of core antenna and the peripheral antenna (2). In oxygenic photosynthetic organisms, the core antennae of photosystem (PS)² II and PSI are composed of CP43/37 and P700-chlorophyll (Chl) a-protein complexes (CP1), respectively. These complexes contain Chl a and -carotene, which function as photosynthetic pigments (1, 3). The notable features of these core antennae are their highly conserved compositions of pigments and the composition of their proteins, which do not change under any environmental conditions. Even if the organisms synthesize other pigments such as Chl b or xanthophylls, these pigments are not incorporated into the core antenna complexes. On the other hand, peripheral antennae are highly diverse among photosynthetic organisms (4). Cyanobacteria and red algae contain phycobilisome as a peripheral antenna (5), one that is composed of open tetrapyrrole pigments. However, brown algae (6) and dinoflagellates (7) possess fucoxanthin-Chl a/c-protein complexes and peridinin-Chl a-protein complexes, respectively. Both of these organisms use carotenoids as the major light-harvesting pigments for photosynthesis. Although both prochlorophytes and green plants have Chl b in their peripheral antenna complexes, the structures of these complexes are quite different from one another. Chl b is bound to prochlorophytes-Chl b-binding protein in prochlorophytes (8). On the other hand, in green plants, Chl b is bound to light-harvesting Chl a/b-protein complexes (LHC) (9), which belong to the LHC superfamily (6). Thus, the pigment distribution is strictly controlled among core and peripheral antenna complexes.

The diverse pigment composition of peripheral antenna complexes is a beneficial feature that enables plants to absorb multiple wavelengths from the broad range of the light spectrum that is available for photosynthesis. Chl a harvests light energy in the blue and red region. However, pigments of peripheral antenna complexes are capable of absorbing different light spectra that cannot be absorbed by the core antenna. For example, Chl b absorbs light at around 470 and 650 nm (10, 11) and fucoxanthin at 550 nm (12), and phycobilins absorb light between 470 and 570 nm (13). In addition to expanding the range of absorption spectra, the peripheral antennae are also capable of regulating the amount of absorbed light energy by changing photosynthetic antenna size (14, 15) or by state transition (16).

Although the aforementioned studies have greatly advanced our understanding of pigment function, they have a shortcoming in that they do not explain why all the pigments, except for Chl a and -carotene, are excluded from the core complexes. The first question concerning the pigment systems in green plants is how the distribution of different Chls among plural complexes is controlled; in particular, how is Chl b preferentially incorporated into LHCs? The next logical question to ask is why the core complex must contain only Chl a but not Chl b.

Chl b is synthesized by chlorophyllide a oxygenase (CAO) in both green plants and prochlorophytes (17–19). According to the hypothesis of Hoober and Eggink (20), CAO associates with LHCII apoproteins on chloroplast envelopes, and the chlorophyllide b that is synthesized by CAO is directly transferred to LHCII apoproteins. This direct transfer to the apoprotein enables the preferential incorporation of Chl b into LHCII (20). This idea was supported by an investigation of the domain structure within Arabidopsis CAO (AtCAO) (19). Alignment of amino acid sequences of CAOs from various organisms clarified the presence of a regulatory domain within AtCAO. Prochlirothrix hollandica CAO (PhCAO), however, does not contain the conserved regulatory domain in its sequence. Because of the observation that Prochlorothrix lacks the regulatory domain, it might be reasonable to speculate that it is involved in LHC formation.

If AtCAO is indeed involved in the control of preferential incorporation of Chl b into LHC apoproteins, it would be reasonable to consider that the distribution of Chl b will not properly proceed when AtCAO is replaced by PhCAO in Arabidopsis. In this study, we introduced PhCAO into an Arabidopsis ch1–1 mutant, which contains a defective CAO gene and is therefore unable to synthesize Chl b. As a result of the PhCAO introduction into the mutant background, the Chl b-less phenotype was rescued, and Chl b was incorporated into the core complexes in the transgenic plants (PhCAO plants). In this study, we discuss the control mechanism of Chl b distribution and the photosynthetic performance of these transgenic plants that contain Chl b in their core antenna complexes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plant Materials and Growth Conditions—Arabidopsis thaliana (Columbia ecotype) was grown in a chamber equipped with white fluorescent lamps (FLR40SSW, NEC Co., Ltd., Tokyo, Japan) under a 16-h photoperiod at a light intensity of 80 µE m^–2 s^–1 at 23 °C. For high light experiments, 25-day-old Arabidopsis plants were transferred from the low light conditions mentioned above to high light conditions. The intensity of light was incrementally increased for 4 days (1 day of 200 µE/m² s + 1 day of 300 µE/m² s + 1 day of 400 µE/m² s + 1 day of 700 µE). These plants were grown for an additional 4 days at the light intensity of 1000 µE m^–2 s^–1 and then used for the pigment determination and fluorescence measurement.

For the genetic transformation of Arabidopsis, 1–168 bp of the AtCAO coding region (as the transit peptide) and the full coding region for PhCAO were subcloned into the pGreenII vector (21). We incorporated the cauliflower mosaic virus 35S promoter and the tobacco mosaic virus sequence (kindly provided by Dr. Niwa, University of Shizuoka). In the following order, the constructs contained the 35S promoter, the sequence (22), the AtCAO transit peptide, the PhCAO coding region, and the nopaline synthase terminator. The resulting plasmids were introduced into Agrobacterium tumefaciens, and the homozygous Arabidopsis chlorina ch1–1 mutant line plant background was transformed.

Pigment Determination—Pigments from the leaves were extracted in acetone, and the extract was centrifuged at 10,000 x g for 5 min, and the supernatant was loaded onto a C18 column (YMC AL303 250 x 4.6 mm, 5 µm, YMC Co., Ltd., Kyoto, Japan). The sample was eluted with an isocratic flow of solvent A (100% methanol) for 17 min, followed by solvent B (60% methanol, 20% ethanol, 20% hexane) for 8 min at a flow rate of 1.2 ml/min. For -carotene determination, the supernatant was separated with an isocratic flow of solvent B for 20 min at a flow rate of 1.0 ml/min. The eluates were monitored by an SPD-10AV spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan) at a wavelength of 450 nm. For the Chl determination of Chl-protein complexes, samples were separated on a C18 column (CLC-ODS, 150 x 6.0 mm, Shimadzu Co., Kyoto, Japan) with an isocratic flow of 100% methanol.

Purification of Envelope and Thylakoid Membranes from Chloroplasts—Envelope and thylakoid membranes were isolated from chloroplasts as described previously (23). Ten g of the leaves from Arabidopsis were homogenized in a blender with 250 ml of ice-cold buffer containing 0.45 M sorbitol, 20 mM Tricine/NaOH (pH 8.4), 10 mM NaHCO₃, 10 mM EDTA, and 0.1% bovine serum albumin. A chloroplast pellet was obtained by centrifugation at 1500 x g for 3 min. The chloroplasts were disrupted in 10 mM MOPS (pH 7.6), 4 mM MgCl₂, and 1 mM phenylmethylsulfonyl fluoride. Chloroplast subfractions were separated on a step gradient of 2.0, 0.93, and 0.6 M sucrose in buffer R (10 mM MOPS (pH 7.6), and 4 mM MgCl₂) by ultrcentrifugation at 70,000 x g for 1 h. The chloroplast envelope fraction collected at the interface between 0.6 and 0.93 M sucrose layers and the thylakoid fraction were collected at the interface between 0.93 and 2.0 M sucrose layers.

Isolation and Spectral Measurement of PSI, LHC, and BBY Particles—Thylakoid membranes were solubilized in 0.8% Triton X-100 to a final concentration of 0.8 mg Chl/ml and centrifuged at 10,000 x g for 10 min. The green supernatant was loaded onto a linear 0.1–0.8 M sucrose density gradient containing 0.08% Triton X-100 and centrifuged at 40,000 rpm in a Hitachi RP50 ultracentrifuge for 14 h at 4 °C. A green band, which corresponded to LHCII, was collected from the sucrose density gradient, and LHCII was purified by adding MgCl₂ and KCl (24). PSI particles were obtained as a nonfluorescent green pellet (25). BBY PSII particles were isolated by the combination of Triton X-100 and centrifugation (26). Absorption spectra of these particles were measured at room temperatures using a Hitachi U-3310 spectrophotometer.

Gel Electrophoresis of Proteins and Pigment-Protein Complexes—Proteins that were isolated from thylakoid membranes corresponding to 10 µg of Chl were suspended in Laemmli buffer and separated on slab gels containing 14% polyacrylamide. Gels were subsequently stained with Coomassie Blue for visualization of protein bands.

Thylakoid membranes were solubilized in 0.5% SDS. Chl-protein complexes were separated by native green gel electrophoresis according to the method of Anderson et al. (10).

Preparation of Thylakoid Membranes—Rosette leaves were homogenized with a razor blade blender in an isolation buffer containing 50 mM Tricine-NaOH (pH 8.0), 350 mM sucrose, and 5 mM EDTA. The homogenate was filtered through a nylon mesh and centrifuged at 10,000 x g for 10 min. The resultant pellet (crude chloroplasts) was resuspended in 5 mM EDTA (pH 8.0) and centrifuged at 10,000 x g for 10 min. The pellet was resuspended in water at a concentration of 2 mg of Chl/ml.

Immunoblot Analysis—Total protein was extracted from leaves by grinding with extraction buffer (50 mM Tris (pH 6.8), 10% (w/v) glycerol, 2% (w/v) SDS, and 6% (v/v) 2-mercaptoerhanol), and these samples were centrifuged at 10,000 x g for 5 min. The supernatants were separated by 14% polyacrylamide (containing 6 M urea) SDS-PAGE, and the resolved proteins were transferred onto Hybond-P membrane (Amersham Biosciences). Proteins were labeled with anti-CP1 or anti-CP43 or anti-LHCII rabbit antibodies. Anti-rabbit IgG linked to horseradish peroxidase was used as secondary antibodies. Chemiluminescent detection was performed using ECL plus Western blotting detection system (Amersham Biosciences) according to the manufacturer's instructions. For determination of the PhCAO localization, 1.0 µg of stroma, envelope, and thylakoid fractions were separated by 10% SDS-PAGE and labeled with antibodies against AtCAO, Tic110 of pea (kindly provided by Dr. Nakai, Osaka University) or LHCII.

Electron Microscopy—Five-week-old Arabidopsis leaves were harvested and soaked with primary fixation buffer (0.05 M PIPES, 0.1 M sucrose, 1% paraformaldehyde, 2.5% glutaraldehyde (pH 7.4)) and post-fixed for 1.5 h in secondary fixation buffer (1% OsO₄ in 100 mM cacodylate buffer (pH 7.4)). Specimens were subsequently stained and observed as described previously (27).

Gas Exchange Measurement—Determinations of CO₂ exchange were made using a portable computerized open system IRGA (LI-6400, Li-Cor, Lincoln, NE). Gas exchange was measured on rosette leaves at 20 °C. The leaves were placed across the short dimension of a 2 x 3-cm leaf cuvette, and they were exposed to LED light at an intensity of 70 or 1000 µmol photons m^–2 s^–1 and to 0.036% CO₂ in the cuvette. The temperature was maintained at 20 °C during all measurements.

Fluorescence Measurement—Maximal photochemical efficiency of PSII (Fv/Fm) was measured using a PAM 2000 fluorometer (H. Waltz, Effeltrich, Germany). Rosette leaves were dark-adapted for 5 min and Fv/Fm was measured at 20 °C. This 5-min dark treatment resulted in the complete oxidization of Q_A.

State Transitions—State transitions were also measured by using a PAM 2000 fluorometer (H. Waltz, Effeltrich, Germany) as described previously (28, 29). State transitions were induced by PSII light (100 µEm^–2 s^–1 blue light ( = 375–595 nm) from a lamp equipped with a Corning 4-96 filter) and PSI light (far-red light provided by PAM 2000 fluorometer). Maximal fluorescence yield in state 1 (Fm1) and in state 2 (Fm2) was then determined. State transitions were calculated as Fm1/Fm2.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rescue of the Chl b-less Phenotype by Prochlorothrix CAO—Although Chl b is synthesized by CAO in both green plants and prochlorophytes, the structures of CAO proteins are quite different (Fig. 1A). Mature AtCAO is predicted to contain 501 amino acids and three separate domains that are designated A-, B-, and C-domains. The C-domain possesses a Rieske center and non-heme iron-binding sites and is capable of catalyzing the conversion Chl a to Chl b by itself (19, 23). The A-domain is highly conserved in higher plants and is predicted to have a control function. Although the lengths of the B-domains are highly conserved between green plants, they do not exhibit sequence similarity. Despite the occurrence of some similarities as mentioned above, the overall structure of PhCAO is much different from AtCAO; specifically, PhCAO is smaller and it consists of a sequence corresponding to the C-domain and a small C-terminal extension. It was proposed that CAO interacts with LHC proteins on the chloroplast envelope and delivers chlorophyllide b to LHC (20). Based on this hypothesis, the construction of LHC and core complexes might be disturbed if AtCAO is replaced by PhCAO in Arabidopsis.

To replace the endogenous CAO by PhCAO, we introduced PhCAO into the Arabidopsis ch1–1 mutant that has a defective CAO gene and shows a Chl b-less phenotype (30). The Chl b-less phenotype was rescued by the introduction of PhCAO. This observation indicates that PhCAO is capable of catalyzing Chl b synthesis in higher plants, although the structure of PhCAO is much different from that of AtCAO. Furthermore, the Chl a/b ratio in the transgenic plants (PhCAO plants) (Fig. 1B) was decreased to 1.1, which was extremely low compared with 3.0–4.0 in the wild type. It is important to note that a ratio this low has never been reported in any previous experiments to the best of our knowledge.

We determined the localization of PhCAO in chloroplasts by immunoblot analysis using anti-AtCAO antibodies (Fig. 1C). Chloroplasts were isolated and fractionated by sucrose density gradient ultracentrifugation. AtCAO could not be detected in any fraction from wild type (data not shown), because accumulation of endogenous CAO was below detectable levels by immunoblot analysis (23). On the other hand, PhCAO was detected in thylakoid membrane fraction at the predicted molecular size, indicating that PhCAO protein was primarily localized in thylakoid membranes in PhCAO plants.

Spectral Characteristics of Photosynthetic Particles—There are three possible explanations for the low Chl a/b ratio that was observed in PhCAO plants. The first explanation is that a large amount of LHCII accumulated in chloroplasts. However, this low Chl a/b ratio could not be achieved by the increase in LHC levels. This conclusion was reached because reported Chl a/b ratios of LHCII are 1.2–1.4 (1, 11), and those of minor LHCs are higher than 1.2 (1, 13). The second explanation is that free Chl b accumulated in PhCAO plants. However, it is well known that free Chl generates reactive oxygen species and induces cell death. PhCAO plants are able to grow under light conditions (Fig. 1B), indicating that free Chl b does not accumulate in PhCAO plants. We subsequently hypothesized that the organization of pigment molecules are altered in PhCAO plants. As a means to test this hypothesis, we determined absorbance spectra and Chl a/b ratios of PSI, LHCII, and PSII particles (BBY particles) in PhCAO plants (Fig. 2). In comparison with the wild type, the Chl a/b ratios of these three samples were extremely low in PhCAO plants (Table 1). Specifically, the absorbance around 650 nm had increased, and absorbance within the long wavelength region (between 680 and 700 nm) had decreased. The wavelength of absorption maximum in PSI particles from the wild type was 680 nm, but measurements from PhCAO plants detected an absorption maximum at 678 nm (Fig. 2A). Although the absorption maximum of PSII particles and LHCII did not change in PhCAO plants, the absorbance corresponding to Chl a (676 nm) had decreased with the concomitant increase in the absorbance that corresponded to Chl b (650 nm) (Fig. 2, B and C). Chl a/b ratios of the three particles are shown in Table 1. Consistent with previous reports, the Chl a/b ratio of PSI was higher (8.09) than that of PSII particles (1.85) and that of LHCII (1.31) in wild type plants. However, in PhCAO plants, Chl a/b ratios of these three particles were drastically reduced (Table 1). These results suggest that a large amount of Chl b was incorporated in PSI, PSII, and LHCII in PhCAO plants compared with wild type plants. To our knowledge, the low Chl a/b ratios that are reported in this study have not been observed in previous experiments. We observed no fluorescence emitted from Chl b with these three samples (data not shown), indicating the high efficiency of energy transfer from Chl b to Chl a in these complexes.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Introduction of PhCAO gene into Arabidopsis Chl b-less mutant. A, the domain structures of CAO of A. thaliana and P. hollandica. AtCAO consists of a transit peptide (TP) and three (A–C) domains. PhCAO only contains the C-domain and a C-terminal extension of 31 amino acids (a.a.). The C-domain is a catalytic domain for converting Chl a to Chl b. B, phenotypes of PhCAO plants. PhCAO gene was introduced into Arabidopsis ch1–1 as described under "Experimental Procedures." It is important to note that although the Chl a/b ratio of PhCAO plants was very low compared with wild type, PhCAO plants can grow well under the low light conditions. C, localization of PhCAO in chloroplasts. Stroma, envelope, and thylakoid membranes were isolated from chloroplasts of PhCAO plants as described under "Experimental Procedures." An equal amount of proteins from these fractions were subjected to immunoblot analyses using anti-CAO, anti-Tic110, or anti-LHCII antibodies. S, stroma; E, envelope; T, thylakoids. Tic110 and LHCII were used as an envelope marker and a thylakoid marker, respectively.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Spectral properties of pigment-protein complexes in wild type and PhCAO plants. Absorbance spectra of PSI particles, LHCII, and BBY (PSII) particles isolated from wild type (WT) Columbia and PhCAO plants. PSI and LHCII were isolated by sucrose density gradient centrifugation, and BBY particles were isolated by the combination of Triton X-100 and centrifugation.

View larger version (59K): [in this window] [in a new window]   FIGURE 3. Chl-protein complexes separated by native green gel electrophoresis. Thylakoid membranes of wild type (WT) and PhCAO plants were prepared, and Chl-protein complexes were separated by native green gel electrophoresis as described under "Experimental Procedures." Chl-protein complexes corresponding to the bands are presented on the left side of the gel. CP1*; CP1-LHCI complexes; LHCP1, LHCP2, and LHCP3, monomeric, dimeric, and trimeric LHCP, respectively. FC, free Chls.

View larger version (52K): [in this window] [in a new window]   FIGURE 4. Protein compositions of thylakoid membranes in wild type and PhCAO plants. A, thylakoid membranes from wild type (WT) and PhCAO plants were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (see "Experimental Procedures"). B, immunoblot analyses of Chl-binding proteins. Leaf extracts corresponding to 50 µgof rosette leaves (which were also corresponding to 0.12 nmol of Chls in both samples) from wild type and PhCAO plants were subjected to SDS-PAGE, and CP1, CP43, and LHCII were detected by using anti-CP1, anti-CP43, and anti-LHCII antibodies.

View larger version (103K): [in this window] [in a new window]   FIGURE 5. Transmission electron microscopy of chloroplast structure. A, wild type; B, PhCAO plants. Thylakoid membranes and grana stacking of PhCAO chloroplasts were developed like those of wild type plants. Bars = 1 µm.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Growth rates of wild type and PhCAO plants. The fresh weights of the aerial parts were measured. There were no significant differences between wild type (WT) and PhCAO plants. Vertical bars represent S.D. (n = 6).

We then investigated the CO₂ exchange rate under low light (70 µE/m² s) and high light (1000 µE/m² s) conditions. In wild type plants, the CO₂ exchange rate was 2.72 mol/m² s under low light, and it increased to 5.85 mol/m s under high light (Table 4). There were no significant differences in the CO₂ exchange rates between wild type and PhCAO plants.

View larger version (157K): [in this window] [in a new window]   FIGURE 7. Effects of high light treatment on wild type and PhCAO plants. Wild type (A and C) and PhCAO plants (B and D) were grown under the low light (A and B) or high light condition (C and D) as described under "Experimental Procedures."

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Growth under the high light conditions. Wild type (WT) and PhCAO plants were grown under the low light conditions and then transferred to high light conditions. Light intensities were incrementally increased to 1000 µE/m2 s over 4 days so that plants were acclimated to high light conditions. Plants were then grown under the light intensity of 1000 µE/m2 s for additional 4 days. The fresh weights of rosette leaves (A) and total fresh weights of the aerial parts (B) were measured at 0, 4, and 8 days of high light treatment. Vertical bars represent S.D. (n = 8).

We next investigated the photosynthetic performance under the low and high light conditions by pulse-modulated fluorescence methods. In wild type plants, the Fv/Fm ratio was 0.82 under low light conditions, and it decreased to 0.63 under high light conditions. The Fv/Fm ratio of PhCAO was 0.73 under low light conditions, but it decreased drastically to 0.19 under high light conditions (Table 6). These data indicate that the PSII electron transport in PhCAO plants was slightly less efficient than that of wild type under low light conditions, but PSII in PhCAO plants was photoinhibited under high light conditions.

View larger version (33K): [in this window] [in a new window]   FIGURE 9. Carotenoid contents in wild type and PhCAO plants under the low light and high light condition. Carotenoid contents in wild type (WT) and PhCAO plants under the low light (LL) and high light (HL) conditions were analyzed as described under "Experimental Procedures." Neo, neoxanthin; Vio, violaxanthin; Ant, antheraxanthin; Zea, zeaxanthin; Lut, lutein; -car, -carotene. Vertical bars represent S.D. (n = 5).

View larger version (17K): [in this window] [in a new window]   FIGURE 10. Induction of qN in wild type and PhCAO plants. Fluorescence during induction of qN was measured with a PAM fluorometer as described under "Experimental Procedures." qN was calculated from the fluorescence data (data not shown) as 1 – Fv'/Fv. Vertical bars represent S.D. (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Distribution of Chls a and b Is Not Determined by the Binding Affinity—In green plants, LHCs are the peripheral antenna complexes that contain Chl a and b, but the core antennae of the PSI and PSII are considered to be the Chl a-protein complexes (1). There are two possible mechanisms for the specific localization of Chl b in LHC. The first proposed scenario is that Chl b has a high affinity for LHC, but it lacks affinity for the core complexes. We reported previously that when cyanobacteria acquired AtCAO in their genome, Chl b was synthesized, and P700-Chl a-protein complexes were functionally converted to Chl a/b-protein complexes (42). Furthermore, when AtCAO was introduced into a PSI-less mutant of cyanobacteria, Chl b replaced part of Chl a in the PSII core, and the energy absorbed by Chl b was efficiently transferred to the PSII reaction centers (43). These results strongly indicate that both core complexes have the capacity to bind Chl b in cyanobacteria. In the present study, we showed that when PhCAO was introduced into a Chl b-less mutant of Arabidopsis, Chl b was actively synthesized and incorporated into the core antenna complexes. The Chl a/b ratios of CP1 and CPa were 1.5 in PhCAO plants. This value is similar to that of Chl b-binding complexes such as LHCs. This indicates that core antenna complexes have a high affinity to Chl b. Considering these results, the preferential distribution of Chl b into LHC is not achieved by binding affinity.

The second possibility is that some control mechanisms are required for the preferential distribution of Chl b to LHC. In this study, we showed that the selective distribution of Chl b was disturbed when endogenous CAO was replaced with PhCAO. This result strongly suggests that CAO is involved in the distribution of Chl in green plants. Hoober and Eggink (20) proposed an hypothesis for the formation of LHC in which a direct interaction between LHC and CAO was postulated. In this proposed mechanism, CAO interacts with LHC apoproteins, and chlorophyllide a in LHC apoproteins is used as the substrate of CAO. Newly synthesized chlorophyllide b is transferred to LHC apoproteins. The interaction between LHC and CAO was also proposed (43) based on the finding that the synthesis of Chl b in cyanobacterium, which had the AtCAO gene in its genome, is stimulated by co-expression of the Lhcb gene. These interactions are not expected with PhCAO, because Prochlorothrix contains prochlorophytes-Chl b-binding protein as Chl b-binding proteins (44) instead of the LHC, which is found in green plants. If the plants have PhCAO instead of their endogenous CAO, the LHC could not interact with PhCAO, and Chl b would be distributed among various apoproteins only by their affinities. This distribution of Chl b would result in its incorporation into core complexes.

If CAO is involved in the controlled distribution of Chl b, the next step is to determine what protein moiety interacts with LHC. We reported previously that higher plant CAO consisted of three domains, A–C. The C-domain has a catalytic function and can convert Chl a to Chl b by itself. The A-domain regulates the stability of CAO proteins (23), and it is possible that the B-domain functions as a linker by connecting the adjoining A- and C-domains. PhCAO contains a sequence that corresponds to the catalytic C-domain and a small C-terminal extension of 31 amino acids. Considering these results, the A-domain is the most plausible candidate for the interaction with LHC, which enables Chl b to distribute into LHC. This conclusion was reached because the A-domain is a unique structure of CAOs in LHC-containing organisms. The other possibility is that the C-domain is involved in the control of Chl distribution because considerable differences in the amino acid sequences of catalytic domain are observed between AtCAO and PhCAO. Although the mechanisms of the assembly of the pigments with the proteins are not completely understood at this time, studies on CAO could provide a clue to understanding the assembly of the pigment-protein complexes. However, we could not exclude the possibility that the distribution of Chls cannot be controlled properly when the ratio of Chl a/b is beyond the threshold, because Chl a/b ratio was extremely low in PhCAO plants.

Prochlorococcus is a marine prokaryote that has divinyl Chl a and b (44). The divinyl Chl a/b ratio of isolated PSI particles from Prochlorococcus is 3.1; however, these PSI particles possess low amounts of peripheral antenna complexes (45). If divinyl Chl b does not exist in the core complexes, this low amount of peripheral antenna complexes cannot account for the divinyl Chl b in PSI particles. Based on these results, the authors discussed the possibility that the core complexes of PSI bind divinyl Chl b in Prochlorococcus. This is consistent with our hypothesis that Chl b can incorporate into core complexes and function as a photosynthetic pigment if there are no regulatory mechanisms for the selective distribution of Chl b to peripheral antenna complexes. Whether selective distribution of Chl a and b among Chl-protein complexes is found only in green plants or in all Chl b-containing organisms should be clarified.

In higher plants, LHCII has been considered to bind a fixed number of Chl a and b molecules, and the Chl a/b ratios were reported to be 1.2–1.4 (11). Consistent with these data, the Chl a/b ratio of trimeric LHCII purified by native SDS-PAGE in wild type plants was 1.2 in this study (Table 2). Recently, the crystal structure of LHCII was solved, and it was shown that individual Chl-binding sites are occupied by either Chl a or b, and no mixed sites are observed (9). However, in our experiments we found that the Chl a/b ratio of LHCII decreased to 0.8 in PhCAO plants, a low value that has not been reported previously with any green plants. The alteration of Chl a/b ratio of LHCII has been reported with in vitro and in vivo experiments. For example, the Chl a/b ratio of isolated LHC from green algae Dunaliella salina was found to change in response to growth irradiance (46, 47). Pigment-protein complexes reconstituted in vitro using recombinant apoproteins also had different Chl a/b ratios depending on the Chl a/b ratios in the reaction mixture. However, the resulting complexes bound the same total number of Chls (48, 49). In this study, we showed that the Chl a/b ratio of LHCII can also change in higher plants. Although these alterations were found in our transgenic analysis, it is still not clear whether the Chl a/b ratio of LHCII changes under natural conditions in higher plants. The physiological significance of the flexibility of the Chl a/b ratio of LHCII in higher plants is an interesting area that warrants further investigation.

Photosynthetic Performance of Chl b-containing Core Complexes—Except for Acaryochloris marina, which has Chl d in core complexes (50), all the core complexes of oxygenic photosynthetic organisms contain only Chl a as the Chl species; however, the reason for this exclusivity remains completely unknown. To address this question, we investigated the photosynthetic performance of photosystems in which 40% of Chl a in the core complexes was replaced by Chl b. The resultant transgenic plants that possessed low Chl a/b ratios were able to grow photoautotrophically; this is the first study to document that the presence of Chl b in the core complexes does not prevent photosynthesis. Absorbance spectra of PSI, LHCII, and PSII (BBY particles) from PhCAO plants have increased absorbance corresponding to Chl b. Fluorescence spectra were also changed by incorporation of Chl b (data not shown). Although these spectral characteristics were altered, the fluorescence of Chl b was not observed with thylakoid or isolated particles under room or liquid N₂ temperatures, suggesting that Chl b in core complexes harvests light energy and efficiently transfers it to neighboring Chl a. Excitation spectra monitored at 735 and 690 nm fluorescence derived from PSI and PSII, respectively, also showed that Chl b in core complexes functioned as a photosynthetic pigment (data not shown). Although Chl b efficiently transferred light energy to Chl a in PhCAO plants, alterations of energy transfer among pigments are expected. Physical measurements such as time-resolved analysis of Chl fluorescence are required for the detailed analysis of energy transfer.

When the plants were grown under the low light conditions, there were no significant differences in CO₂ exchanging activity between wild type and PhCAO plants. The growth rates of PhCAO plants were almost the same as those of the wild type, which is consistent with CO₂ exchange rates. Collectively, these data indicate that Chl a can be replaced by Chl b in the core complexes without significant alterations of photosynthetic performances under the low light conditions. At present, we are unable to find any reason why Chl b was excluded from the core complexes.

Besides the physical aspects, the discrimination of Chl molecules by different apoproteins might be necessary for the control of the accumulation of LHCII, a phenomenon that is directly involved in the regulation of antenna size. It has been proposed that LHCII accumulation is regulated by the synthesis of Chl b through the stabilization of LHCII in the thylakoid membranes (20). This mechanism is realized only when Chl b is preferentially incorporated into LHCII, otherwise Chl b synthesis is not coupled with LHCII formation.

Fv/Fm ratios of PhCAO plants decreased by 20% of wild type plants under the low light conditions. However, the ratios were drastically decreased under the high light conditions in PhCAO plants. We determined that the thermal dissipation of excess light energy functioned in Chl b-rich PSII particles, because qN appeared similarly after transfer to high light in both wild type and PhCAO plants. The question then arose as to why the PSII was damaged under high light conditions. One possible mechanism is as follows: the conformational changes of PSII complexes are induced by Chl b, resulting in the damage of PSII. Conformational changes in PSI particles by the presence of Chl b was reported previously in cyanobacteria (51) where a trimeric form of cyanobacterial PSI particles was converted to a monomeric PSI by introducing Chl b into PSI complexes. The second possible mechanism to explain the damage to PSII includes the inhibition of the D1 repair cycle. Under high light conditions, rapid D1 turnover is required because the D1 protein is damaged under high light conditions (52). In transgenic plants, Chl b interferes with the assembly of Chl a with D1 proteins during the repair cycle because the distribution of Chl b is not controlled. Further studies are required to clarify the nature of the photodamage affecting PSII in PhCAO plants.

	FOOTNOTES

 
^* This work is supported in part by Grant-in-aid 15370015 (to A. T.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute of Low Temperature Science, Hokkaido University, Kita-ku, N19 W8, Sapporo, 060-0819 Japan. Tel./Fax: 81-11-706-5493; E-mail: ayumi{at}pop.lowtem.hokudai.ac.jp.

² The abbreviations used are: PS, photosystem; LHC, light-harvesting chlorophyll; CAO, chlorophyllide a oxygenase; Chl, chlorophyll; MOPS, 3-(N-morpholino)propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; AtCAO, Arabidopsis thaliana CAO; PhCAO, P. hollandica CAO; HPLC, high pressure liquid chromatography.

	ACKNOWLEDGMENTS

 
We thank Dr. Yasuo Niwa (University of Shizuoka, Japan) for providing the 35SrsGFP vector, Drs. Roger Hellens and Phil Mullineaux (John Innes Centre, UK) for providing the pGreenII vector kit, and Dr. Masato Nakai for providing anti-Tic110 antibodies. We also thank Dr. Megumi Moriya for excellent technical assistance in electron microscopy.

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