Clp protease controls chlorophyll b synthesis by regulating the level of chlorophyllide a oxygenase

Chlorophyll b is one of the major light-harvesting pigments in green plants and it is essential for optimal light harvesting. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO) which consists of A, B and C domains. Previously, we demonstrated that the C domain alone has a catalytic function, while the A and B domains control the level of CAO protein in response to chlorophyll b accumulation. We hypothesized that the accumulation of chlorophyll b triggers the proteolytic degradation of CAO. In this study, in order to gain further insight into this regulatory mechanism we screened for mutants that have defects in the control of CAO accumulation. Seeds from a transgenic line of Arabidopsis which overexpressed a CAO-GFP fusion were mutagenized and their progenies were screened by laser-scanning confocal microscopy for mutants showing an elevated level of GFP fluorescence. One particular mutant (dca1) exhibited stronger GFP fluorescence and accumulated a GFP-CAO fusion protein at a higher level. Concomitantly, the chlorophyll a to b ratio decreased in this mutant. The mutation in the dca1 mutant was mapped to the ClpC1 gene, thereby indicating that a chloroplast Clp protease is involved in regulating chlorophyll b biosynthesis through the destabilization of CAO protein in response to the accumulation of chlorophyll b.     

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.     

Characterization of Arabidopsis mutants defective in the regulation of chlorophyllide a oxygenase

Chlorophyll b is one of the major light-harvesting pigments produced by land plants, green algae and several cyanobacterial species. It is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which in higher plants consists of three domains, namely, A, B, and C. We previously demonstrated that the C domain exhibits a catalytic function, whereas the A domain destabilizes the CAO protein in the presence of chlorophyll b, thus regulating the cellular level of CAO. In a previous study, we also presented genetic evidence demonstrating the involvement of Clp protease in the destabilization of CAO. In this study, in order to gain further insight into the regulatory mechanism of CAO, we screened for mutants defective in the control of CAO accumulation. Seeds from an Arabidopsis transgenic plant overexpressing a chimeric protein consisting of the A and B domains of CAO and green fluorescent protein (GFP) were mutagenized by ethyl methane sulfonate. We screened the progenies of the transgenic plants by laser-scanning confocal microscopy, and isolated a total of 66 mutants exhibiting significant GFP fluorescence. By immunoblotting analysis, we confirmed that these mutants accumulated the fusion protein of the N-terminal domains of CAO and GFP at a high level. We further divided these mutants into seven groups by distribution patterns of the fusion protein, and characterized them by pigment and immunoblotting analyses. Based on these analyses, we proposed a model to describe the regulatory mechanism of CAO.     

Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.     

Chlorophyll b-Deficient Mutants of Rice: II. Antenna Chlorophyll a/b-Proteins of Photosystem I and II

Chlorophyll-protein complexes of the wild type and 16 strainsof chlorina mutants of rice were investigated by gel electrophoresis.An antenna chlorophyll a/b-protein of photosystem II (LHC-II)was present in reduced amounts in Type II chlorina mutants whichhave the chlorophyll a/b ratios of 10–15, and was totallyabsent from Type I chlorina mutants which lack chlorophyll b.Another antenna chlorophyll-protein of photosystem I (LHC-I)containing two polypeptides of 20 and 21 kDa was also presentin the Type II mutants but not in the Type I mutants. The polypeptideprofiles of the thylakoid membranes indicate that Type I mutantslack both the 20 and 21 kDa polypeptides, whereas the abundanceof the two polypeptides relative to the CPI apoprotein in theType II mutants is comparable with that in the wild type. Itis concluded that the 20 and 21 kDa polypeptides are both relatedto LHC-I and are normally synthesized and accumulated in theType II mutants. (Received June 6, 1985; Accepted August 6, 1985)     

Pigment shuffling in antenna systems achieved by expressing prokaryotic chlorophyllide a oxygenase in Arabidopsis

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), approximately 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.     

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.     

Chlorophyll b expressed in Cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins

Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophyll a oxygenase) cDNA was introduced into the genome of Synechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyll b content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophyll a-protein complex (CP1). Furthermore, chlorophyll b in CP1 transferred light energy to chlorophyll a, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of approximately 4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper.     

Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

浅绿叶水稻突变体的特性与遗传分析

在簇生稻与粳稻日本晴杂交后代F8世代中发现一个能稳定遗传的浅绿叶色突变体(pgl,pale green leaf)。与野生型相比,突变体pgl株高、剑叶宽、主穗粒数和千粒重均显著下降。从幼苗开始,突变体pgl叶片都表现为浅绿色。在苗期和抽穗期突变体叶片的叶绿素含量都极显著低于野生型,其中叶绿素b的含量极低,仅为0.002~0.003 mg/g,突变体pgl表现为叶绿素b的缺失。在分蘖期与齐穗期,突变体pgl的净光合作用速率与野生型相当。叶绿体超微结构观察表明突变体pgl的叶绿体基质片层和堆叠层数较少。遗传分析发现浅绿叶色表型由一对隐性细胞核基因控制。采用BSA法,通过全基因组SNP芯片分析,浅绿叶色基因pgl被定位于水稻第10染色体上的22806614~23000408区间,与R1022900951CA标记紧密连锁。突变体pgl与另外3个浅绿叶色突变体(W1、Y406和Y45)的等位性检测结果表明浅绿叶色基因pgl与突变体W1的浅绿叶色基因为等位基因。对pgl的候选基因LOC_Os10g41780(叶绿素a加氧酶,chlorophyll a oxygenase)的序列比对发现,在突变体pgl中,LOC_Os10g41780在第2507和3136位碱基处分别发生1个T的缺失和T变成C的替换。分析发现,第3136位碱基位于第9外显子内,其碱基T变C的替换导致其编码的精氨酸变成色氨酸。本研究鉴定的突变体pgl和W1为LOC_Os10g41780的新变异,为阐明浅绿叶色形成的分子机理和光合作用机理的研究提供了特异资源。     

The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F(0) fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17+/-0.017 ns in the chlorina Photosystem II core to a value within the range 0.42+/-0.036-0.47+/-0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalized system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.     

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting.

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.     

Determination of a Chloroplast Degron in the Regulatory Domain of Chlorophyllide a Oxygenase

Chlorophyll b is one of the major photosynthetic pigments of plants. The regulation of chlorophyll b biosynthesis is important for plants in order to acclimate to changing environmental conditions. In the chloroplast, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), a Rieske-type monooxygenase. The activity of this enzyme is regulated at the level of protein stability via a feedback mechanism through chlorophyll b. The Clp protease and the N-terminal domain (designated the A domain) of CAO are essential for the regulatory mechanism. In this study, we aimed to identify the specific amino acid residue or the sequence within the A domain that is essential for this regulation. To accomplish this goal, we randomly introduced base substitutions into the A domain and searched for potentially important residues by analyzing 1,000 transformants of Arabidopsis thaliana. However, none of the single amino acid substitutions significantly stabilized CAO. Therefore, we generated serial deletions in the A domain and expressed these deletions in the background of CAO-deficient Arabidopsis mutant. We found that the amino acid sequence ⁹⁷QDLLTIMILH¹⁰⁶ is essential for the regulation of the protein stability. We furthermore determined that this sequence induces the destabilization of green fluorescent protein. These results suggest that this sequence serves as a degradation signal that is recognized by proteases functioning in the chloroplast.