In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Anthocyanins act as a sugar-buffer and an alternative electron sink in response to starch depletion during leaf senescence: a case study on a typical anthocyanic tree species, Acer japonicum.

We hypothesized that anthocyanins act as a sugar-buffer and an alternative electron sink during leaf senescence to prevent sugar-mediated early senescence and photoinhibition. To elucidate the role of anthocyanin, we monitored seasonal changes in photosynthetic traits, sugar, starch and N contents, pigment composition, and gene expression profiles in leaves exposed to substantially different light conditions within a canopy of an adult fullmoon maple (Acer japonicum) tree. Enhancement of starch amylolysis accompanied by cessation of starch synthesis occurred in the same manner independent of light conditions. Leaf sugar contents increased, but reached upper limits in the late stage of leaf senescence, even though leaf anthocyanins further increased after complete depletion of starch. Sun-exposed leaves maintained higher energy consumption via electron flow than shade-grown leaves during leaf N resorption. Thus, anthocyanins accumulated in sun-exposed leaves might have a regulative role as a sugar-buffer, retarding leaf senescence, and an indirect photoprotective role as an alternative sink for electron consumption to compensate declines in other metabolic processes such as starch and protein synthesis. In this context, anthocyanins may be key substrates protecting both outer-canopy leaves (against photoinhibition) and inner-canopy leaves (via shading by outer-canopy leaves) from high light stress during N resorption.

Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach.

Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.

Reversible down-regulation of photosystems I and II leads to fast photosynthesis recovery after long-term drought in Jatropha curcas.

Jatropha curcas is a drought-tolerant plant that maintains its photosynthetic pigments under prolonged drought, and quickly regains its photosynthetic capacity when water is available. It has been reported that drought stress leads to increased thermal dissipation in PSII, but that of PSI has been barely investigated, perhaps due to technical limitations in measuring the PSI absolute quantum yield. In this study, we combined biochemical analysis and spectroscopic measurements using an integrating sphere, and verified that the quantum yields of both photosystems are temporarily down-regulated under drought. We found that the decrease in the quantum yield of PSII was accompanied by a decrease in the core complexes of PSII while light-harvesting complexes are maintained under drought. In addition, in drought-treated plants, we observed a decrease in the absolute quantum yield of PSI as compared with the well-watered control, while the amount of PSI did not change, indicating that non-photochemical quenching occurs in PSI. The down-regulation of both photosystems was quickly lifted in a few days upon re-watering. Our results indicate, that in J. curcas under drought, the down-regulation of both PSII and PSI quantum yield protects the photosynthetic machinery from uncontrolled photodamage.

Dynamic seasonal changes in photosynthesis systems in leaves of Asarum tamaense, an evergreen understorey herbaceous species.

BACKGROUND AND AIMS: Evergreen herbaceous species in the deciduous forest understorey maintain their photosystems in long-lived leaves under dynamic seasonal changes in light and temperature. However, in evergreen understorey herbs, it is unknown how photosynthetic electron transport acclimates to seasonal changes in forest understorey environments, and what photoprotection systems function in excess energy dissipation under high-light and low-temperature environments in winter. METHODS: Here, we used Asarum tamaense, an evergreen herbaceous species in the deciduous forest understorey with a single-flush and long-lived leaves, and measured photosynthetic CO2 assimilation and electron transport in leaves throughout the year. The contents of photosynthetic proteins, pigments and primary metabolites were determined from regularly collected leaves. KEY RESULTS: Both the rates of CO2 assimilation and electron transport under saturated light were kept low in summer, but increased in autumn and winter in A. tamaense leaves. Although the contents of photosynthetic proteins including Rubisco did not increase in autumn and winter, the proton motive force and ΔpH across the thylakoid membrane were high in summer and decreased from summer to winter to a great extent. These decreases alleviated the suppression by lumen acidification and increased the electron transport rate in winter. The content and composition of carotenoids changed seasonally, which may affect changes in non-photochemical quenching from summer to winter. Winter leaves accumulated proline and malate, which may support cold acclimation. CONCLUSIONS: In A. tamaense leaves, the increase in photosynthetic electron transport rates in winter was not due to an increase in photosynthetic enzyme contents, but due to the activation of photosynthetic enzymes and/or release of limitation of photosynthetic electron flow. These seasonal changes in the regulation of electron transport and also the changes in several photoprotection systems should support the acclimation of photosynthetic C gain under dynamic environmental changes throughout the year.

Heterologous complementation systems verify the mosaic distribution of three distinct protoporphyrinogen IX oxidase in the cyanobacterial phylum.

The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.

BRZ-INSENSITIVE-PALE GREEN 1 is encoded by chlorophyll biosynthesis enzyme gene that functions in the downstream of brassinosteroid signaling.

Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana.

The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.

Unique Peripheral Antennas in the Photosystems of the Streptophyte Alga Mesostigma viride.

Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI-PSII megacomplex, PSII-LHCII, two PSI-LHCI-LHCIIs, PSI-LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI-LHCI-LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII-LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI-LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Distribution and functional analysis of the two types of 8-vinyl reductase involved in chlorophyll biosynthesis in marine cyanobacteria.

In the chlorophyll biosynthesis pathway, the 8-vinyl group of the chlorophyll precursor is reduced to an ethyl group by 8-vinyl reductase. Two isozymes of 8-vinyl reductase have been described in oxygenic photosynthetic organisms: one encoded by BciA and another by BciB. Only BciB contains an [Fe-S] cluster and most cyanobacteria harbor this form; whereas a few contain BciA. Given this disparity in distribution, cyanobacterial BciA has remained largely overlooked, which has limited understanding of chlorophyll biosynthesis in these microorganisms. Here, we reveal that cyanobacterial BciA encodes a functional 8-vinyl reductase, as evidenced by measuring the in vitro activity of recombinant Synechococcus and Acaryochloris BciA. Genomic comparison revealed that BciB had been replaced by BciA during evolution of the marine cyanobacterium Synechococcus, and coincided with replacement of Fe-superoxide dismutase (SOD) with Ni-SOD. These findings imply that the acquisition of BciA confers an adaptive advantage to cyanobacteria living in low-iron oceanic environments.

Horizontal Transfer of Promiscuous Activity from Nonphotosynthetic Bacteria Contributed to Evolution of Chlorophyll Degradation Pathway.

The relationship between enzymes and substrates does not perfectly match the "lock and key" model, because enzymes act on molecules other than their true substrate in different catalytic reactions. Such biologically nonfunctional reactions are called "promiscuous activities." Promiscuous activities are apparently useless, but they can be an important starting point for enzyme evolution. It has been hypothesized that enzymes with low promiscuous activity will show enhanced promiscuous activity under selection pressure and become new specialists through gene duplication. Although this is the prevailing scenario, there are two major problems: 1) it would not apply to prokaryotes because horizontal gene transfer is more significant than gene duplication and 2) there is no direct evidence that promiscuous activity is low without selection pressure. We propose a new scenario including various levels of promiscuous activity throughout a clade and horizontal gene transfer. STAY-GREEN (SGR), a chlorophyll a-Mg dechelating enzyme, has homologous genes in bacteria lacking chlorophyll. We found that some bacterial SGR homologs have much higher Mg-dechelating activities than those of green plant SGRs, while others have no activity, indicating that the level of promiscuous activity varies. A phylogenetic analysis suggests that a bacterial SGR homolog with high dechelating activity was horizontally transferred to a photosynthetic eukaryote. Some SGR homologs acted on various chlorophyll molecules that are not used as substrates by green plant SGRs, indicating that SGR acquired substrate specificity after transfer to eukaryotes. We propose that horizontal transfer of high promiscuous activity is one process of new enzyme acquisition.

In Vitro Enzymatic Activity Assays Implicate the Existence of the Chlorophyll Cycle in Chlorophyll b-Containing Cyanobacteria.

In plants, chlorophyll (Chl) a and b are interconvertible by the action of three enzymes-chlorophyllide a oxygenase, Chl b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR). These reactions are collectively referred to as the Chl cycle. In plants, this cyclic pathway ubiquitously exists and plays essential roles in acclimation to different light conditions at various developmental stages. By contrast, only a limited number of cyanobacteria species produce Chl b, and these include Prochlorococcus, Prochloron, Prochlorothrix and Acaryochloris. In this study, we investigated a possible existence of the Chl cycle in Chl b synthesizing cyanobacteria by testing in vitro enzymatic activities of CBR and HCAR homologs from Prochlorothrix hollandica and Acaryochloris RCC1774. All of these proteins show respective CBR and HCAR activity in vitro, indicating that both cyanobacteria possess the potential to complete the Chl cycle. It is also found that CBR and HCAR orthologs are distributed only in the Chl b-containing cyanobacteria that habitat shallow seas or freshwater, where light conditions change dynamically, whereas they are not found in Prochlorococcus species that usually habitat environments with fixed lighting. Taken together, our results implicate a possibility that the Chl cycle functions for light acclimation in Chl b-containing cyanobacteria.

Formation of a PSI-PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens.

Mosses are one of the earliest land plants that diverged from fresh-water green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.

The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC2 D is the major, and possibly sole scaffold in plastids.

The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling.

Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.

Deficiency of the Stroma-Lamellar Protein LIL8/PSB33 Affects Energy Transfer Around PSI in Arabidopsis.

Light-harvesting-like (LIL) proteins are a group of proteins that share a consensus amino acid sequence with light-harvesting Chl-binding (LHC) proteins. We hypothesized that they might be involved in photosynthesis-related processes. In order to gain a better understanding of a potential role in photosynthesis-related processes, we examined the most recently identified LIL protein, LIL8/PSB33. Recently, it was suggested that this protein is an auxiliary PSII core protein which is involved in organization of the PSII supercomplex. However, we found that the majority of LIL8/PSB33 was localized in stroma lamellae, where PSI is predominant. Moreover, the PSI antenna sizes measured under visible light were slightly increased in the lil8 mutants which lack LIL8/PSB33 protein. Analysis of fluorescence decay kinetics and fluorescence decay-associated spectra indicated that energy transfer to quenching sites within PSI was partially hampered in these mutants. On the other hand, analysis of the steady-state fluorescence spectra in these mutants indicates that a population of LHCII is energetically disconnected from PSII. Taken together, we suggest that LIL8/PSB33 is involved in the fine-tuning of light harvesting and/or energy transfer around both photosystems.

Reexamination of chlorophyllase function implies its involvement in defense against chewing herbivores.

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.