Loss of the N-terminal domain of chlorophyllide aoxygenase induces photodamage during greening of Arabidopsis seedlings
© Yamasato et al; licensee BioMed Central Ltd. 2008
Received: 05 November 2007
Accepted: 12 June 2008
Published: 12 June 2008
Chlorophyll b is a major photosynthetic pigment in green plants that is synthesized by chlorophyllide a oxygenase (CAO). The regulation of chlorophyll b biosynthesis is an important determinant for the antenna size of photosystems. Chlorophyll b synthesis is partly regulated on a transcriptional level by the expression of the CAO gene. In addition, the synthesis of chlorophyll b is strictly regulated on a protein level by the stability of the CAO enzyme. CAO consists of three domains, which are sequentially named from the N terminus as the A, B and C domains. The A domain of CAO participates in the regulation of the CAO protein stability.
In order to clarify the physiological function of the A domain, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plants which either overexpressed the complete CAO or a truncated version of CAO lacking the A domain. The transgenic plants overexpressing the A-domain-deleted CAO accumulated an excess amount of chlorophyll b during greening. The transgenic plants which lacked the A domain either died or were obviously retarded when they were exposed to continuous light immediately after etiolation. In addition, the loss of the A domain in CAO impaired another step of chlorophyll biosynthesis, namely the conversion of divinyl-protochlorophyllide a to monovinyl protochlorophyllide a under dark conditions.
The A domain of CAO regulates the level of CAO, and thus prevents the excess accumulation of chlorophyll b. This function of the A domain is especially important during the greening stage of etiolated seedlings. At this stage, the plants are vulnerable to photodamages which could be caused by excessive chlorophyll b accumulation. In addition, de-regulation of the CAO level affects monovinyl-protochlorophyllide biosynthesis in darkness by unknown mechanisms. In conclusion, the A domain of CAO is essential in the control of chlorophyll biosynthesis and in the survival of seedlings during de-etiolation especially under strong illumination.
Chlorophyll molecules are major components of photosynthesis and play essential roles in harvesting light energy and charge separation. Chlorophylls are actively synthesized during greening and are assembled with various apoproteins to form chlorophyll-protein complexes. Apoproteins of core antenna complexes solely bind chlorophyll a, while apoproteins of peripheral antenna complexes bind both chlorophyll a and chlorophyll b . Since these chlorophyll-protein complexes are stoichiometrically assembled to form functional photosystems, chlorophyll metabolism must be tightly regulated to coordinate with the formation of these complexes. Previous reports have suggested that the regulation of chlorophyll metabolisms is important from the viewpoint of photodamage. Most of the chlorophyll intermediates are photoreactive compounds . If the regulation of 5-aminolevulinic acid (ALA), a precursor of tetrapyrrole synthesis, is defective as the result of mutation of a regulation factor, protochlorophyllide is accumulated in excessive amounts under dark conditions . Consequently, this overabundance of protochlorophyllide results in the generation of reactive oxygen species (ROS) and finally causes growth retardation or cell death during greening .
At the last step of chlorophyll biosynthesis, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO). Since an increase in CAO mRNA levels stimulates chlorophyll b synthesis, CAO activity is considered to be partly regulated by CAO gene expression [5–7]. In addition to the regulation on the transcriptional level, chlorophyll b synthesis is also regulated by protein stability. CAO consists of three domains, which are named sequentially from the N terminus as the A, B and C domains . The C domain contains binding motifs for a Rieske center and non-heme iron and catalyzes the conversion of chlorophyll a to chlorophyll b [4, 8, 9]. Although the precise function of the B domain is not known, it is considered to possibly function as a linker between the A and C domains . Although the A domain is not involved in catalytic function, it regulates CAO protein stability [8, 11]. When the A domain was removed from CAO, and only BC domains were introduced and overexpressed in Arabidopsis (Arabidopsis thaliana), the protein levels of CAO were drastically increased and chlorophyll b was excessively accumulated . We recently determined that the chloroplast Clp protease plays an important role in CAO protein stability . However, excess chlorophyll b accumulation does not result in a distinct alteration of photosynthetic activity and does not induce photodamage in green leaves . Therefore, at the present time it is still not known why the protein level of CAO is strictly regulated by the A domain.
In order to answer this question, we introduced genes corresponding to either the complete CAO sequence or BC domains into Arabidopsis. We subsequently investigated the greening processes of these transgenic plants under various light conditions. Interestingly, transgenic plants expressing only the BC domains died when the etiolated seedlings were exposed to continuous light. In addition, chlorophyll synthesis was disturbed in these plants during etiolation and greening of seedlings. We discuss the role of the A domain in the regulation of chlorophyll synthesis and chloroplast development.
CAO without the A domain caused photodamage during greening
As shown in Figs. 1 and 2, our transgenic lines did not show any obvious phenotypical differences between the lines. In the subsequent experiments described within this study, we also examined all transgenic lines shown in Figs. 1 and 2. All of the lines produced results that were nearly identical.
Loss of the A domain results in a perturbation of chlorophyll metabolism
Total protochlorophyllide accumulation in etiolated seedlings.
Total protochlorophyllide/etiolated seedling (pmole)
Divinyl-protochlorophyllide/total protochlorophyllide (molar ratio)
0.78 ± 0.02
8.09 ± 1.98
0.37 ± 0.03
0.33 ± 0.01
2.56 ± 0.56
0.80 ± 0.05
1.01 ± 0.09
6.29 ± 2.08
0.32 ± 0.03
Total protochlorophyllide accumulation in etiolated seedlings during dark incubation.
Total protochlorophyllide/etiolated seedling (pmole)
Dark incubation after light treatment
0.70 ± 0.01
0.77 ± 0.16
0.49 ± 0.01
0.25 ± 0.02
0.68 ± 0.13
1.28 ± 0.01
The A domain protects cells against photodamage during greening
We previously reported on the light sensitivity of transgenic Arabidopsis plants overexpressing prokaryotic CAO which does not have the A domain. These transgenic plants accumulated much more chlorophyll b than tGBCch plants . In this previous report, we concluded that the excess accumulation of chlorophyll b did not cause photodamage in the fully greened leaves of the transgenic plants . However, in this study, we found that most of the etiolated tGBCch seedlings died during greening not only under high light conditions, but also under low light conditions. Without the presence of chlorophyll a, chlorophyll b would not be incorporated into apoproteins. As a consequence, the free chlorophyll b might generate ROS . In contrast to tGBCch, transgenic plants overexpressing the full-length CAO accumulated both chlorophyll a and chlorophyll b immediately after the onset of illumination. Unlike tGBCch plants, the full-length-CAO overexpression lines did not exhibit photodamage. It is possible that chlorophyll b is assembled with apoproteins in the presence of chlorophyll a. As a result, the generation of ROS might have been suppressed. Collectively, these results indicate that the A domain is essential in preventing photodamages that could be caused by excess chlorophyll b accumulation during greening. The etiolated seedlings of the transgenic Arabidopsis overexpressing prokaryotic CAO might cause obvious photodamage.
An unusually high level of 7-hydroxymethyl chlorophyll a, an intermediate molecule in the conversion between chlorophyll b to chlorophyll a, accumulated in the green leaves of tGBCch (Fig. 8). This observation might indicate that excess chlorophyll b is reconverted to chlorophyll a by the chlorophyll cycle . It is not likely that 7-hydroxymethyl chlorophyll was formed in the course of the chlorophyll a to chlorophyll b conversion. Since CAO catalyzes the two-step reaction from chlorophyll a to chlorophyll b by itself, the intermediate molecule, 7-hydroxymethyl chlorophyll, is supposed to be associated with the CAO enzyme and is not released during the reaction . In our study, 7-hydroxymethyl chlorophyll was not found just after the start of the illumination of etiolated seedlings, although chlorophyll b was actively synthesized (Fig. 7). Therefore, it would be reasonable to assume that 7-hydroxymethyl chlorophyll was predominantly formed in the course of the chlorophyll b to chlorophyll a conversion.
Accumulation of GFP-BC protein interferes with the regulation of chlorophyll biosynthesis
In comparison to wild type plants, chlorophyll accumulation during greening was suppressed in tGBCch seedlings, even under low light conditions. It is possible that this suppression might be due to the oxidative stress generated by free chlorophyll b. The overabundance of protochlorophyllide generates ROS, which causes growth retardation or cell death during greening in the flu mutant . Recently, we found that the chlorophyll biosynthesis pathway is a target of oxidative stress . However, all of the phenotypes of tGBCch could not be explained by oxidative stress alone. For example, the level of protochlorophyllide a in etiolated tissues of tGBCch was lower than that of wild type. Since these plants were not exposed to light, the involvement of ROS in the inhibition of protochlorophyllide accumulation can be excluded. We also observed an increase in the ratio of divinyl-protochlorophyllide to the total protochlorophyllide in tGBCch transgenic plants. These data indicate that the accumulation of the BC domains of CAO inhibits the activity of 3, 8-divinyl protochlorophyllide a 8-vinyl reductase. On the contrary, overexpression of the GFP-ABC protein enhanced chlorophyll accumulation. These phenomena are very interesting in terms of biological regulation since both CAO and truncated CAO affected not only chlorophyll b synthesis but also other chlorophyll metabolic steps. These results are reasonable because some enzymes, which are related to chlorophyll biosynthesis, are reported to form complexes or interact with other enzymes. For example, glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase form a complex which enables an efficient substrate trafficking between two enzymes . In addition, BchH, one of the three subunits of Mg chelatase of Rhodobacter capsulatus, accelerated the Mg-protoporphyrin IX monomethyl transferase activity . If CAO does indeed interact with other enzymes, the accumulation of truncated CAO could potentially interfere with the activity of the target enzymes.
In addition, the loss of the A domain in CAO impaired the conversion of divinyl-protochlorophyllide a to monovinyl protochlorophyllide a, and obviously reduced total protochlorophyllide a level under dark conditions. A possible scenario for the reduction of protochlorophyllide accumulation in the tGBCch seedlings is that the excessively-accumulated CAO in these seedlings interfered with the enzyme that catalyzes the formation of monovinyl-protochlorophyllide a from divinyl-protochlorophyllide a, the latter of which may inhibit an earlier step of chlorophyll biosynthesis in a feedback manner. Further studies are necessary and warranted to clarify this question.
CAO does not catalyze the conversion of protochlorophyllide a to protochlorophyllide b
When etiolated seedlings of wild type are exposed to continuous light, chlorophyll a immediately accumulates after the onset of illumination. Subsequent to illumination, chlorophyll a and b levels gradually increase after several hours of a lag phase. Conversely, in tGBCch plants, chlorophyll b accumulated but chlorophyll a was not detected after 5 min of illumination (Fig. 7). Considering that protochlorophyllide b was not found in etiolated seedlings, these data indicate that chlorophyll a was immediately converted to chlorophyll b in tGBCch seedlings during the early phase of greening (Fig. 9).
Several research groups report that protochlorophyllide b does not exist in etioplasts [20, 21]. Contrary to these reports, another group claimed that the protochlorophyllide b could be detected in etiolated seedlings when they use diethylpyrrocarbonate-containing acetone for the extraction of pigments . It was also proposed that protochlorophyllide b is synthesized via a pathway that is independent from the CAO protein . In addition, a cyanobacteria transformant with the CAO and Lhcb transgenes, accumulated protochlorophyllide b . This observation indicates that CAO is capable of converting protochlorophyllide a to protochlorophyllide b. In this study, we used 100% acetone as an extraction solvent for chlorophyll intermediates in order to avoid the artificial formation of chlorophyll derivatives and to maximize our extraction of the chlorophyll intermediates from seedlings . Consequently, we did not detect any protochlorophyllide b in etiolated tGBCch seedlings, even though a large amount of the CAO protein accumulated. These results indicate that protochlorophyllide b is not a major intermediate product for chlorophyll b synthesis in planta.
Taken together, our results demonstrated that excessive accumulation of chlorophyll b is harmful to plants. This deleterious effect likely disturbs the proper synthesis of chlorophyll intermediates and the accumulation of photosynthetic proteins. Therefore, we conclude that the regulation of the CAO protein by the A domain is essential for the survival of etiolated seedlings under light conditions. Since the A domain functions to maintain proper chlorophyll b levels throughout the development of plants , it would be reasonable to assume that the regulation of the CAO protein level is essential in various aspects of plant development and acclimation to light conditions. Further studies on the regulatory mechanism of CAO may elucidate the physiological roles of this mechanism throughout the life cycle of plants.
Plant materials and growth conditions
The Arabidopsis CAO protein consists of three domains, which are respectively named: A (V37-L170), B (P171-G200) and C (A201-G537) domains . In this study, we used Arabidopsis thaliana Columbia wild type and the ch1-1 mutant which contains a deletion in the CAO gene . In addition, we also used two types of transgenic plants overexpressing chimeric fusions of the CAO protein and the GFP. The two Arabidopsis transformants, tGBCch and tGABCch, expressed fusion transgenes corresponding to transit peptide-GFP-B-C domains and transit peptide-GFP-A-B-C domains in the ch1-1 background, respectively . Three homozygous lines for each transgenic plant tGBCch and tGABCch, were used in this study. All of the transgenic plants were isolated independently. Arabidopsis seedlings were grown at 22°C on agar plates (0.7% [w/v]) containing 1/2 diluted Murashige-Skoog medium. Plants were grown under low light (70 μE m-2 s-1 from fluorescent bulbs) or high light (300 μE m-2 s-1 from xenon light bulbs [3.6 kW] equipped with ND filters, TGE-2S3, Tabai, Osaka, Japan) conditions.
Reverse transcriptase PCR
Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 200 etiolated seedlings which were grown under dark conditions for 4 days. RNA was extracted from etiolated seedlings after a period of greening for 24 hours. One μg of isolated RNA was reverse-transcribed into cDNA using a SuperScript III Kit (Invitrogen, Carlsbad, CA). PCR was performed to determine gene expression level using the synthesized first-stranded cDNA sample in a 25 μl reaction. PCR conditions were as follows: 96°C (2 min); 25 cycles of 96°C (30 sec), 55°C (30 min), 72°C (1 min). The amount of cDNA template for the PCR was normalized by β-tubulin (TUB2) gene expression level. The primers used for RT-PCR were as follows: For TUB2, 5'-CTC AAG AGG TTC TCA GCA GTA-3' and 5'-TCA CCT TCT TCA TCC GCA GTT-3'; For CAO, 5'-AAC GAG GGA CGT ATT CAA TGT CCG-3' and 5'-AGA AGA AGG TAA ACA GAC ATG G-3'; For GFP, 5'-ATG GTG AGC AAG GGC GAG G-3' and 5'-TTA CTT GTA CAG CTC GTC CA-3'.
Ten seedlings were homogenized with 100 μl of extraction buffer (50 mM Tris [pH 6.8], 2 mM EDTA, 10% [w/v] glycerol, 2% [w/v] SDS, 6% [v/v] 2-mercaptoethanol). The isolated supernatants (25 μl) were subsequently subjected to SDS-PAGE. The resolved proteins were blotted onto a hybond-P membrane (GE Healthcare, Buckinghamshire, UK). A 1/5000-diluted anti-GFP (Invitrogen) antibody, a 1/2000-diluted anti-CAO antibody  and a 1/5000-diluted anti-Lhcb  rabbit antibody were used to detect specific proteins as previously described . Cross-reactive protein bands were developed using anti-rabbit IgG that was linked to horseradish peroxidase (GE Healthcare). An ECL plus western blotting analysis kit was use for the chemiluminescent detection of antigens (GE Healthcare).
Fluorescence images were recorded on an Axioplan fluorescence microscope (× 20 objective lens, Carl Zeiss, Jena, Germany) which was integrated in an MRC 1024 confocal laser-scanning microscopic system (Bio-rad laboratories, Hercules, CA). Samples were excited by an argon laser (25 mW) at 488 nm and the GFP and chlorophyll fluorescence were recorded at 522 and 680 nm, respectively. The images were processed with the Adobe Photoshop 4.0 software (Adobe, San Joze, CA).
Chlorophylls were initially extracted from the seedlings with 100% acetone and the extracts were subsequently diluted to 80% acetone with water. Diluted extracts were then subjected to HPLC analysis (Shim-pack CLC-ODS column, 6.0 × 150 mm; Shimadzu, Kyoto, Japan) using methanol as the elution agent at a flow rate of 1.7 ml min-1. The compositions of chlorophyll species were calculated from chromatographic peak profiles at 650 nm .
Protochlorophyllides extracted from the seedlings were analyzed by HPLC on a Symmetry C8 column (4.6 × 150 mm; Waters Corporation, MA, USA) according to the previously described method of Zapata et al. . Elution profiles and the spectra of the eluted pigments were recorded continuously in the range of 400 to 700 nm by SPD-M10AAV (Shimadzu). The compositions of protochlorophyllide were calculated from chromatographic peak profiles at 440 nm .
This work was supported by a Grant-in-Aid for Creative Scientific Research (No. 17GS0314 to AT) and Grants-in-Aid for Scientific Research (No. 18770025 to AY; No. 68700307 to RT) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We would like to thank Drs. R. Hellens and P. Mullineaux (John Innes Centre, UK) for kindly providing the pGreen II plasmids. We would also like to thank Dr. Y. Niwa (Division of Food Science, University of Shizuoka, Japan) for providing us with the sGFP (S65T) plasmid.
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