Fluorescent Chlorophyll Catabolite Research Articles

Comparative genomics analysis of pheophorbide a oxygenase (PAO) genes in eight pyrus genomes and their regulatory role in multiple stress responses in Chinese pear (Pyrus bretschneideri).

Pyrus (pear) is among the most nutritious fruits and contains fibers that have great health benefits to humans. It is mostly cultivated in temperate regions globally and is highly subjected to biotic and abiotic stresses which affect its yield. Pheophorbide a oxygenase (PAO) is an essential component of the chlorophyll degradation system and contributes to the senescence of leaves. It is responsible for opening the pheophorbide a porphyrin macrocycle and forming the main fluorescent chlorophyll catabolite However, this gene family and its members have not been explored in Pyrus genomes. Here we report a pangenome-wide investigation has been conducted on eight Pyrus genomes: Cuiguan, Shanxi Duli, Zhongai 1, Nijisseiki, Yunhong No.1, d'Anjou, Bartlett v2.0, and Dangshansuli v.1.1. The phylogenetic history, their gene structure, conservation patterns of motifs, their distribution on chromosomes, and gene duplication are studied in detail which shows the intraspecific structural conservation as well as evolutionary patterns of Pyrus PAOs. Cis-elements, protein-protein interactions (PPI), and the Gene Ontology (GO) enrichment analyses show their potential biological functions. Furthermore, their expression in various tissues, fruit hardening conditions, and drought stress conditions is also studied. Based on phylogenetics, the identified PAOs were divided into four groups. The expansion of this gene family in Pyrus is caused by both tandem and segmental duplication. Moreover, positive and negative selection pressure equally directed the gene's duplication process. The Pyrus PAO genes were enriched in hormones-related, light, development, and stress-related elements. RNA-seq data analysis showed that PAOs have varied levels of expression under diseased and abiotic stress conditions. The 3D structures of PAOs are also predicted to get more insights into functional conservation. Our research can be used further to get a deeper knowledge of the PAO gene family in Pyrus and to guide future research on improving the genetic composition of Pyrus to enhance stress tolerance.

Postharvest senescent dark spot development mechanism of Musa acuminata ("Khai" banana) peel associated with chlorophyll degradation and stomata cell death.

The occurrence of the postharvest physiological disorder of dark spots on the peel of the ripened "Khai" banana has led to a reduction in its commercial value. The objective of the present study was to investigate the development mechanisms of senescence dark spots of the "Khai" (Musa AA group) banana peel in relation to chlorophyll degradation and stomata cell death. Freshly harvested bananas (commercial mature green stage) were let to ripened at 25±2°C (90%-95% RH). Peel color, senescent spots, DNA degradation, chlorophyll content, chlorophyll-degrading enzyme activities were assessed. The senescent dark spots developed on the ripened bananas right after 6days of storage, which coincided with remarkably increased DNA degradation, and a rapid decreased of hue angle value and total chlorophyll content which indicated the chlorophyll degradation. The activities of chlorophyllase, chlorophyll-degrading peroxidase and pheophytinase increased gradually to the highest point where the chlorophyll content drastically reduced and the appearance of the dark spots was first recorded after 6days of storage. These dark spots were observed to be surrounded with a bright luminescent ring of hypermodified fluorescent chlorophyll catabolites (FCCs), the product of chlorophyll breakdown. Additionally, the scanning electron microscope (SEM) revealed that the dark spots were found to have originated from the collapsed cells around the stomata of the ripened banana peel whereby the chlorophyll was entirely diminished. PRACTICAL APPLICATIONS: This research revealed the senescent dark spot development mechanisms of the "Khai" banana peel. The dark spot development symptom on the banana peel surface was caused by the senescence and cell death of the relevant stomata, further associated with chlorophyll degradation. Therefore, any further research into minimizing the dark spot symptom must focus on preventing or delaying stomata senescence and cell death.

Profile of Chlorophyll Catabolites in Senescent Leaves of Epipremnun aureum Includes a Catabolite Esterified with Hydroxytyrosol 1-O-Glucoside.

Despite the fact that chlorophyll degradation is a physiological phenomenon occurring daily in all photosynthetic tissues, chlorophyll catabolites are not fully identified. Three new forms (1, 3, and 4) of linear chlorophyll catabolites (phyllobilins) have been characterized in senescent leaves of Epipremnun aureum with spectroscopic data. Compound 1 is a hypermodified blue fluorescent chlorophyll catabolite (hmFCC) esterified with the potent antioxidant hydroxytyrosol. The sequestration of this phenol by a chlorophyll catabolite could explain the physiological meaning of the persistence of hmFCCs in some senescent plants. Compound 3, a yellow chlorophyll catabolite (YCC) originated from the oxidation at C-15 of 1. YCCs have been identified previously and are exclusively formed in the plant vacuole from the final nonfluorescent chlorophyll catabolites (NCCs). The presence of 3 in leaves implies a new reaction in chlorophyll catabolism, as the characterization of 3 implies that YCCs can be also be oxidized in the cytosol from FCCs. Finally, phyllobilin 4 represents a new type of YCC characterized by an inflexible bicyclo glucosyl moiety linked through an intramolecular esterification of the propionic acid residue with the C-3 hydroxy group. The corresponding NCC precursor was recently identified and now the characterization of 4 shows that even this rigid structure can be further oxidized. Undoubtedly, the characterization of phyllobilins is essential to completely comprehend chlorophyll degradation.

The fluorescent blue glow of banana fruits is not due to symplasmic plastidial catabolism but arises from insoluble phenols estherified to the cell wall

Banana fruits are firstly green due to chlorophyll, then yellow due to carotenoids and finally turn black due to polyphenols. However, bananas glow blue when observed under UV light. It has been reported that chlorophylls fade to give rise to fluorescent chlorophyll catabolites (FCCs) in senescent banana leaves and in ripening banana peels. FCCs are short lived catabolic intermediates that ultimately lead to non-fluorescent chlorophyll catabolites (NCCs). FCCs are abundant in bananas due to hypermodification; therefore, it was concluded that FCC caused yellow bananas to glow blue. Experiments were performed in order to shed new light into the autofluorescence phenomenon. Microscopy performed on living plant samples contradict the interpretation that the fluorescent blue glow is mainly caused by FCC inside the cell. Blue fluorescence in banana emerges from the cell wall, not from the symplasm. It is not primarily caused by soluble chlorophyll catabolites in the vacuoles or senescing plastids. Insoluble phenolics from the apoplast make bananas shine strongly blue under black light. Chlorophyll is a light trap that generates black holes of blue fluorescence, and therefore cells with chloroplasts glow less blue. The white pulp of banana fruits shine more strongly than the outer peel. In both tissues autofluorescence arises from insoluble phenols that are estherified to the cell wall. In monocot species (banana, maize, sugarcanne), blue fluorescense was strongest in the cell wall, whereas in dicots (e.g. arabidopsis, spearmint, hibiscus), blue fluorescence may be dominant from cytosolic, vacuolar or plastidial compartments.

Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts.

Hydroxylation of chlorophyll catabolites at the so-called C32 position ( Hauenstein et al., 2016 ) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the substrate epi-pFCC (epi-primary Fluorescent Chlorophyll Catabolite) ( Mühlecker et al., 2000 ) to epi-pFCC-OH.

Chlorophyll catabolism in olive fruits (var. Arbequina and Hojiblanca) during maturation

The central reaction of chlorophyll (chl) breakdown pathway occurring during olive fruits maturation is the cleavage of the macrocycle pheophorbide a to a primary fluorescent chl catabolite (pFCC) and it is catalyzed by two enzymes: pheophorbide a oxygenase (PaO) and red chl catabolite reductase (RCCR). In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). This work demonstrated that RCCR activity of olive fruits is type II. During the study of evolution of PaO and RCCR activities through the olive fruits maturation in two varieties: Hojiblanca and Arbequina, a significant increase in PaO and RCCR activity was found in ripening stage. In addition, the profile and structure of NCCs present in epicarp of this fruit was studied using HPLC/ESI-TOF-MS. Five different NCCs were defined and for the first time the enzymatic reactions implied in chlorophyll degradations in olive fruits elucidated.

Crystal structure of methylesterase family member 16 (MES16) from Arabidopsis thaliana

Methylesterase family member 16 (MES16) is an integral component of chlorophyll breakdown. It catalyzes the demethylation of fluorescent chlorophyll catabolite (FCC) and pheophorbide in vitro, and specifically demethylates FCC in vivo. Here we report the crystal structure of MES16 from Arabidopsis thaliana at 2.8 Å resolution. The structure confirm that MES16 is a member of the α/β-hydrolase superfamily with Ser-87, His-239, and Asp-211 as the catalytic triad. Our biochemical studies reveal that MES16 has esterase activity with methyl-indole acetic acid as the substrate, and the catalytically essential role of Ser-87 has been demonstrated.

Cloning and characterization of the pepper CaPAO gene for defense responses to salt-induced leaf senescence.

BackgroundPheophorbide a oxygenase (PAO) is an important enzyme in the chlorophyll catabolism pathway and is involved in leaf senescence. It opens the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. Previous studies have demonstrated the function of PAO during cell death. However, the characterizaton of PAO during leaf senescence induced by environmental factors is not well understood.MethodsHomology-based cloning and RACE techniques were used to obtain the full-length cDNA of the CaPAO gene. CaPAO expression was determined by quantitative real-time PCR. Function of CaPAO gene were studied using virus-induced gene silencing and transgenic techniques with tobacco plants (Nicotiana tabacum).ResultsA novel PAO gene CaPAO was isolated from pepper (Capsicum annuum L.). The full-length CaPAO cDNA is comprised of 1838 bp, containing an open reading frame of 1614 bp, and encodes a 537 amino acid protein. This deduced protein belongs to the Rieske-type iron-sulfur superfamily, containing a conserved Rieske cluster. CaPAO expression, as determined by quantitative real-time PCR, was higher in leaves than roots, stems and flowers. It was upregulated by abscisic acid, methyl jasmonate and salicylic acid. Moreover, CaPAO was significantly induced by high salinity and osmotic stress treatments and also was regulated by Phytophthora capsici. The virus-induced gene silencing technique was used to silence the CaPAO gene in pepper plants. After 3 days of high salt treatment, the chlorophyll breakdown of CaPAO-silenced pepper plants was retarded. RD29A promoter-inducible expression vector was constructed and transferred into tobacco plant. After 7 days of salt treatment, the leaves of transgenic plants were severely turned into yellow, the lower leaves showed necrotic symptom and chlorophyll content was significantly lower than that in the control plants.ConclusionsThe expression of CaPAO gene was induced in natural senescence and various stresses. The CaPAO gene may be related to defense responses to various stresses and play an important role in salt-induced leaf senescence.

A Dioxobilin-Type Fluorescent Chlorophyll Catabolite as a Transient Early Intermediate of the Dioxobilin-Branch of Chlorophyll Breakdown in Arabidopsis thaliana.

Chlorophyll breakdown in higher plants occurs by the so called “PaO/phyllobilin” path. It generates two major types of phyllobilins, the characteristic 1-formyl-19-oxobilins and the more recently discovered 1,19-dioxobilins. The hypothetical branching point at which the original 1-formyl-19-oxobilins are transformed into 1,19-dioxobilins is still elusive. Here, we clarify this hypothetical crucial transition on the basis of the identification of the first natural 1,19-dioxobilin-type fluorescent chlorophyll catabolite (DFCC). This transient chlorophyll breakdown intermediate was isolated from leaf extracts of Arabidopsis thaliana at an early stage of senescence. The fleetingly existent DFCC was then shown to represent the direct precursor of the major nonfluorescent 1,19-dioxobilin that accumulated in fully senescent leaves.

Hydroxymethylated Dioxobilins in Senescent Arabidopsis thaliana Leaves: Sign of a Puzzling Biosynthetic Intermezzo of Chlorophyll Breakdown.

1-Formyl-19-oxobilin-type tetrapyrroles are characteristic, abundant products of chlorophyll breakdown in senescent leaves. However, in some leaves, 1,19-dioxobilin-type chlorophyll catabolites (DCCs) lacking the formyl group accumulate instead. A P450 enzyme was identified in in vitro studies that removed the formyl group of a primary fluorescent chlorophyll catabolite (pFCC) and generated fluorescent DCCs. These DCCs are precursors of isomeric nonfluorescent DCCs (NDCCs). Here, we report a structural investigation of the NDCCs in senescent leaves of wild-type Arabidopsis thaliana. Four new NDCCs were characterized, two of which carried a stereoselectively added hydroxymethyl group. Such formal DCC hydroxymethylations were previously found in DCCs in leaves of a mutant of A. thaliana. They are now indicated to be a feature of chlorophyll breakdown in A. thaliana, associated with the specific in vivo deformylation of pFCC en route to NDCCs.

Cloning and expression analysis of pepper chlorophyll catabolite reductase gene CaRCCR.

Opening the porphyrin macrocycle of pheophorbide a and forming the primary fluorescent chlorophyll catabolites are key steps in the chlorophyll catabolism pathway. These steps are catalyzed by pheophorbide a oxygenase and red chlorophyll catabolite reductase (RCCR). In this study, a novel RCCR gene, CaRCCR, was isolated from the pepper (Capsicum annuum L.). The full-length CaRCCR complementary DNA is comprised of 1173 bp, contains an open reading frame of 945 bp, and encodes a 314-amino acid protein. This deduced protein belongs to the ferredoxin-dependent bilin reductase family. Amino acid sequence alignment showed that CaRCCR shared a high homology to other higher plant RCCR proteins. CaRCCR expression, as determined by quantitative real-time polymerase chain reaction, was higher in the leaves than the roots, stems, flowers, and immature fruits. CaRCCR expression was almost constant during all phases of leaf development. It was upregulated by abscisic acid, methyl jasmonate, and salicylic acid. Moreover, CaRCCR was induced by high salinity and drought stress treatments; it was also slightly regulated by Phytophthora capsici. Taken together, these results suggest that CaRCCR is involved in defense responses to various stresses.

Photochemical studies of a fluorescent chlorophyll catabolite–source of bright blue fluorescence in plant tissue and efficient sensitizer of singlet oxygen

Fluorescent chlorophyll catabolites (FCCs) are fleeting intermediates of chlorophyll breakdown, which is seen as an enzyme controlled detoxification process of the chlorophylls in plants. However, some plants accumulate large amounts of persistent FCCs, such as in senescent leaves and in peels of yellow bananas. The photophysical properties of such a persistent FCC (Me-sFCC) were investigated in detail. FCCs absorb in the near UV spectral region and show blue fluorescence (max at 437 nm). The Me-sFCC fluorescence had a quantum yield of 0.21 (lifetime 1.6 ns). Photoexcited Me-sFCC intersystem crosses into the triplet state (quantum yield 0.6) and generates efficiently singlet oxygen (quantum yield 0.59). The efficient generation of singlet oxygen makes fluorescent chlorophyll catabolites phototoxic, but might also be useful as a (stress) signal and for defense of the plant tissue against infection by pathogens.

Back Cover: Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles (Chem. Eur. J. 37/2013)

A stunning variety of “hypermodified” blue fluorescent chlorophyll catabolites accumulates in senescent banana leaves, as reported by B. Kräutler et al. in the Full Paper on page 12294 ff. The cover shows a vertical cut through a yellow leaf, as seen by fluorescence microscopy, superimposed with the structures of chlorophyll a and of a major fluorescent catabolite. Colored halos highlight the transformation of red fluorescing chlorophylls to the blue fluorescing breakdown products.

Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles

Chlorophyll breakdown is a visual phenomenon of leaf senescence and fruit ripening. It leads to the formation of colorless chlorophyll catabolites, a group of (chlorophyll-derived bilin-type) linear tetrapyrroles. Here, analysis and structure elucidation of the chlorophyll breakdown products in leaves of banana (Musa acuminata) is reported. In senescent leaves of this monocot all chlorophyll catabolites identified were hypermodified fluorescent chlorophyll catabolites (hmFCCs). Surprisingly, nonfluorescent chlorophyll catabolites (NCCs) were not found, the often abundant and apparently typical final chlorophyll breakdown products in senescent leaves. As a rule, FCCs exist only fleetingly, and they isomerize rapidly to NCCs in the senescent plant cell. Amazingly, in the leaves of banana plants, persistent hmFCCs were identified that accounted for about 80 % of the chlorophyll broken down, and yellow leaves of M. acuminata display a strong blue luminescence. The structures of eight hmFCCs from banana leaves were analyzed by spectroscopic means. The massive accumulation of the hmFCCs in banana leaves, and their functional group characteristics, indicate a chlorophyll breakdown path, the downstream transformations of which are entirely reprogrammed towards the generation of persistent and blue fluorescent FCCs. As expressed earlier in related studies, the present findings call for attention, as to still elusive biological roles of these linear tetrapyrroles.

Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis.

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.

Structures of chlorophyll catabolites in bananas (Musa acuminata) reveal a split path of chlorophyll breakdown in a ripening fruit.

The disappearance of chlorophyll is a visual sign of fruit ripening. Yet, chlorophyll breakdown in fruit has hardly been explored; its non-green degradation products are largely unknown. Here we report the analysis and structure elucidation of colorless tetrapyrrolic chlorophyll breakdown products in commercially available, ripening bananas (Musa acuminata, Cavendish cultivar). In banana peels, chlorophyll catabolites were found in an unprecedented structural richness: a variety of new fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs) were detected. As a rule, FCCs exist only "fleetingly" and are hard to observe. However, in bananas several of the FCCs (named Mc-FCCs) were persistent and carried an ester function at the propionate side-chain. NCCs were less abundant, and exhibited a free propionic acid group, but functional modifications elsewhere. The modifications of NCCs in banana peels were similar to those found in NCCs from senescent leaves. They are presumed to be introduced by enzymatic transformations at the stage of the mostly unobserved, direct FCC-precursors. The observed divergent functional group characteristics of the Mc-FCCs versus those of the Mc-NCCs indicated two major "late" processing lines of chlorophyll breakdown in ripening bananas. The "last common precursor" at the branching point to either the persistent FCCs, or towards the NCCs, was identified as a temporarily abundant "secondary" FCC. The existence of two "downstream" branches of chlorophyll breakdown in banana peels, and the striking accumulation of persistent Mc-FCCs call for attention as to the still-elusive biological roles of the resulting colorless linear tetrapyrroles.

MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis.

During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol.

Knockdown of Os PAO and Os RCCR1 cause different plant death phenotypes in rice

Pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR) catalyze key steps in chlorophyll degradation by opening the porphyrin macrocycle of pheophorbide a and forming the primary non-photoreactive fluorescent chlorophyll catabolite. These genes strongly participate in senescence and reportedly involved in plants’ responses to physical wounding and pathogens. In this report, a single PAO gene ( OsPAO) and two RCCR genes ( OsRCCR1 and OsRCCR2) have been isolated from rice. Expression analysis by semi-quantitative PCR or quantitative real-time PCR showed that OsRCCR1 transcripts were much more abundant than OsRCCR2, and all of these genes were upregulated during senescence and following wound treatment. RNA interference knockdown of OsPAO led to pheophorbide a accumulation in leaves (especially dark-induced senescent leaves) and leaf death from regeneration stage onwards, even transgenic plants inviability after transplantation. While, knockdown of OsRCCR1 resulted in lesion-mimic spots generation in older leaves which died off early in the transgenic plants. These results suggest that OsPAO and OsRCCR1 play key roles in senescence and are involved in wound responses.

A novel blue fluorescent chlorophyll catabolite accumulates in senescent leaves of the peace lily and indicates a split path of chlorophyll breakdown

Colorless, non-fluorescent Chl-catabolites (NCCs) are the typical, ubiquitous products of chlorophyll (Chl)-breakdown in senescent leaves. However, a fluorescent Chl-catabolite (FCC) accumulated in de-greened leaves of Spathiphyllum wallisii (Peace Lily), which showed a weak blue luminescence. The FCC, named Sw-FCC-62, was ‘hypermodified’ with an unprecedented 6-(2-[3,4-dihydroxy-phenyl]-ethyl)-β-glucopyranosidyl ester at the propionyl group. Such esters stabilize FCCs against their typical and rapid, spontaneous isomerization to NCCs. Chl-breakdown in Sp. wallisii thus branches off from the ‘common’ path in leaves, and furnishes unique and ‘persistent’ FCCs. Our findings on ‘hypermodified’ FCCs also call for attention as to possible physiological roles of Chl-catabolites in plants.

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.