Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites,pheophorbide a and pyropheophorbide a,against hepatitis C virus

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo‐Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n‐hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti‐HCV activities with 50%‐inhibitory concentrations (IC₅₀) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity‐guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti‐HCV compound present in the extracts (IC₅₀ = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti‐HCV activity (IC₅₀ = 0.2 μg/mL). The 50%‐cytotoxic concentrations (CC₅₀) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti‐HCV activities. Time‐of‐addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post‐entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.     

The Arabidopsis-accelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a: inhibition of the pheophorbide a oxygenase activity does not lead to the "stay-green" phenotype in Arabidopsis

Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.     

Photocytotoxic pheophorbide-related compounds from Aglaonema simplex

In our screening program for new photosensitizers from the Malaysian biodiversity, we found five pheophorbide-related compounds from the leaves and stems of Aglaonema simplex. Detailed spectroscopic analyses showed that compounds 1-3 and 5 are pheophorbide and hydroxy pheophorbide derivatives of chlorophyll a and b. Compound 4, identified as 15(1)-hydroxypurpurin-7-lactone ethyl methyl diester, was isolated for the first time from the Araceae family. An MTT-based short-term survival assay showed that all five compounds exhibit moderate-to-strong photocytotoxic activities towards human leukemia (HL60) and two oral squamous carcinoma cell lines (HSC-2 and HSC-3). Compounds 4 and 5 showed the strongest photocytotoxicities, with IC(50) values of 0.30-0.41 muM (Table 2). Compounds 1-3 with Et chains at C(17(3)) were less photocytotoxic than the parent pheophorbide a (5).     

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.     

Pheophytin Pheophorbide Hydrolase (Pheophytinase) Is Involved in Chlorophyll Breakdown during Leaf Senescence in Arabidopsis

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.     

Chlorophyll breakdown in higher plants

Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final) breakdown products of chlorophyll was crucial for the recent delineation of a chlorophyll breakdown pathway which is highly conserved in land plants. Pheophorbide a oxygenase is the key enzyme responsible for opening of the chlorin macrocycle of pheophorbide a characteristic to all further breakdown products. Degradation of chlorophyll was rationalized by the need of a senescing cell to detoxify the potentially phototoxic pigment, yet recent investigations in leaves and fruits indicate that chlorophyll catabolites could have physiological roles. This review updates structural information of chlorophyll catabolites and the biochemical reactions involved in their formation, and discusses the significance of chlorophyll breakdown. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

Evolution of Chlorophyll Degradation: The Significance of RCC Reductase

Abstract: In angiosperms the key process of chlorophyll breakdown in senescing leaves is catalyzed by pheophorbide a oxygenase and RCC reductase which, in a metabolically channeled reaction, cleave the porphyrin macrocycle and produce a colourless primary catabolite, pFCC. RCC reductase is responsible for the reduction of the C20/C1 double bond of the intermediary catabolite, RCC. Depending on plant species, RCC reductase produces one of the two C1 stereoisomers, pFCC-1 or pFCC-2. Screening of a large number of taxa for the type of RCCR revealed that the isomer produced is uniform within families. It also revealed that type RCCR-2 is predominant; RCCR-1 seems to represent a recent derivation which in unrelated lineages has evolved independently from RCCR-2. A third type of pFCC was produced by RCCR from basal pteridophytes and some gymnosperms; its structure is unknown. Collectively, the data suggest that the pathway of chlorophyll breakdown is very conserved in vascular plants. RCCR appears to represent a decisive addition to the catabolic pathway: it allows terrestrial plants to metabolize the porphyrin part of the chlorophyll molecule to photodynamically inactive final products that are stored in the vacuoles of senescing mesophyll cells.     

Molecular cloning and function analysis of the stay green gene in rice

Chloroplasts undergo drastic morphological and physiological changes during senescence with a visible symptom of chlorophyll (Chl) degradation. A stay green mutant was identified and then isolated from the japonica rice (Oryza sativa) cv. Huazhiwu by gamma-ray irradiation. The stay green mutant was characterized by Chl retention, stable Chl-protein complexes, and stable thylakoid membrane structures, but lost its photosynthetic competence during senescence. The gene, designated Stay Green Rice (SGR), was cloned by a positional cloning strategy encoding an ancient protein containing a putative chloroplast transit peptide. SGR protein was found in both soluble and thylakoid membranes in rice. SGR, like the gene for pheophorbide a oxygenase (PaO), was constitutively expressed, but was upregulated by dark-induced senescence in rice leaves. Senescence-induced expression of SGR and PaO was enhanced by ABA, but inhibited by cytokinin. Overexpression of SGR reduced the number of lamellae in the grana thylakoids and reduced the Chl content of normally growing leaves. This indicates that upregulation of SGR increases Chl breakdown during senescence in rice. A small quantity of chlorophyllide a accumulated in sgr leaves, but this also accumulated in wild-type rice leaves during senescence. Some pheophorbide a was detected in sgr leaves in the dark. According to these observations, we propose that SGR may be involved in regulating or taking part in the activity of PaO, and then may influence Chl breakdown and degradation of pigment-protein complex.     

Evaluation of two biosynthetic pathways to delta-aminolevulinic acid in Euglena gracilis.

delta-Aminolevulinic acid (ALA), which is an intermediate in the biosynthesis of chlorophyll a, can be biosynthesized via the C5 pathway and the Shemin pathway in Euglena gracilis. Analysis of the (13)C-NMR spectrum of (13)C-labeled methyl pheophorbide a, derived from 13C-labeled chlorophyll a biosynthesized from d-[1-(13)C]glucose by E. gracilis, provided evidence suggesting that ALA incorporated in the (13)C-labeled chlorophyll a was synthesized via both the C5 pathway and the Shemin pathway in a ratio of between 1.5 and 1.7 to one. The methoxyl carbon of the methoxycarbonyl group at C-132 of chlorophyll a was labeled with (13)C. The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3(1), C-P4, C-P6, C-P7(1), C-P8, C-P10, C-P11(1), C-P12, C-P14, C-P15(1) and C-P16.     

Characterization of a virescent chloroplast mutant of tobacco

Virescent mutations produce plants in which young leaves have reduced chlorophyll levels but accumulate nearly normal amounts of chlorophyll as they age; they are predominantly nuclear mutations. We describe here a virescent mutation (designated Vir-c) found in a somatic hybrid line derived from Nicotiana tabacum L. and Nicotiana suaveolens Lehm. This mutation is inherited maternally. Young, half-expanded Vir-c leaves contained three to six times less chlorophyll than did control leaves, and reached maximum chlorophyll levels much later in development. Chlorophyll synthesis rates and chloroplast numbers per cell in Vir-c were similar to the control, and carotenoid content in Vir-c was sufficient to protect chlorophyll from photo-oxidation. Photosynthetic rates of Vir-c at low light intensities suggested a reduced ability to collect light. Electron micrographs of Vir-c chloroplasts from half-expanded leaves showed a significant reduction in thylakoids per granum. The decrease in granal thylakoids was strongly associated with low chlorophyll levels; mature Vir-c leaves with nearly normal chlorophyll content showed normal granal profiles. These results are discussed in relation to virescent mutants previously described.     

Predicting the distribution of Carpinus betulus in Denmark with Ellenberg's Climate Quotient

In this paper we investigate whether Ellenberg's Climate Quotient (EQ, defined as the mean temperature of the warmest month divided by annual precipitation, multiplied by IOOO), can be used to predict the distribution of Carpinus betulus in Denmark. It has been suggested that the competitive relationship between the two tree species Fagus sylvatica and C.betulus is related to EQ in central Europe. Areas with low EQ have dominance of E. sylvatica, whereas higher EQs are associated with dryer and warmer climates that favour C.betulus. To determine if this holds true, also in northern Europe, we investigate the present distribution of C.betulus in Denmark, based on a comprehensive dataset with presence-absence information of the species. We relate the distribution of C.betulus to 12 climate parameters and indices (including EQ), analyse it in a geographical information system and compare the ecology of C.betulus and E sylvatica in four Danish forests, located in different climatic and floristic regions. The highest density of C.betulus was found in eastern Denmark where EQ is high, i.e. summer temperature is relatively high and precipitation low. In the western and south-western parts of the country, where the climate is slightly more wet and more oceanic, there are fewer populations of C.betulus. Based on present climatic data it seems that the climate of Denmark does not limit the occurrence of C.betulus, except perhaps for a small area in western Jutland. We believe that climate changes in the late Holocene cannot alone account for the changes in the distribution of C.befulus in Denmark. And future climate change is likely to affect the distribution of C.betulus with generally better conditions.     

Chlorophyll, Ribulose-1,5-diphosphate Carboxylase, and Hill Reaction Activity in Developing Leaves of Populus deltoides

The synthesis of chlorophyll and ribulose diphosphate carboxylase as well as the development of Hill reaction activity were followed in expanding Populus deltoides leaves and related to photosynthetic patterns. Total chlorophyll, which was not correlated with photosynthetic rate in expanding leaves, decreased slightly with age in very young leaves, due to a decrease in chlorophyll b, but then increased linearly. The ratio of chlorophyll a to b, which rose sharply in young leaves, was highly correlated with the onset of net photosynthesis. Hill reaction activity was very low in young leaves and did not increase significantly until leaves were about half expanded. Ribulose diphosphate carboxylase activity increased in a sigmoid fashion with leaf ontogenesis and closely paralleled development of the photosynthetic system. The study demonstrates the importance of chlorophyll a and Calvin cycle enzyme synthesis to photosynthetic development in expanding leaves.     

Modification of Photosystem I Light Harvesting of Bundle-Sheath Chloroplasts Occurred during the Evolution of NADP-Malic Enzyme C4 Photosynthesis

Low-temperature emission spectra and excitation spectra for chlorophyll fluorescence were recorded from leaves of species of the genus Flaveria (Asteraceae) with C3, C3-C4-intermediate, C4-like, and C4 photosynthesis. Among the latter two groups, high chlorophyll b absorption was observed in excitation spectra for photosystem I (PSI) fluorescence. By comparing leaf data with those from isolated chloroplast fractions, the high chlorophyll b absorption was attributed to the specific properties of the bundle-sheath chloroplasts in leaves from C4 plants. The deconvolution of the PSI excitation spectra and the use of a model revealed that the contribution of photosystem II absorption to the functional antenna of PSI was markedly increased in leaves from three of the five C4-like and C4 species investigated in detail. The two other species exhibited normal, C3-like light-harvesting properties of PSI. The former species are known for efficient carbon assimilation, the latter for decreased efficiencies of carbon assimilation. It is concluded that photosystem II becomes a substantial part of the functional PSI antenna late in the evolution of C4 photosynthesis, and that the composite antenna optimizes the light-harvesting of PSI in bundle-sheath chloroplasts to meet the energy requirements of C4 photosynthesis.     

The turnover of chlorophyll in greening wheat leaves

A method for the estimation of chlorophyll turnover in wheat leaves is presented. This is based on the inhibition of chlorophyll synthesis by treatment of the cut leaves with laevulinic acid (LA), a competitive inhibitor of δ-aminolaevulinic acid dehydratase. The turnover of chlorophyll in young, greening leaves, given short periods of light was a relatively rapid process. However, in seedlings exposed to light for longer periods the turnover became progressively slower, and was measured in days rather than hours.     

Spermine delays leaf senescence in Lactuca sativa and prevents the decay of chloroplast photosystems

Aliphatic polyamines (PAs) are involved in the delay or prevention of plant senescence, but the molecular mechanism is not clarified. The hypothesis is put forward that one of the mechanisms by which PAs modulate leaf senescence and chlorophyll stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by transglutaminase (TGase, R-glutaminylpeptide-amine γ-glutamyltransferase; E.C. 2.3.2.13). The retardation of leaf senescence of Lactuca sativa L. by spermine (Spm) was examined during induced cell death using leaf discs, or during the normal developmental senescence of leaves. Over 3 days, in leaf discs, Spm caused a delay of chlorophyll (Chl) decay, an increase of endogenous TGase activity, and a three-fold increase in chlorophyll content when supplied together with exogenous TGase. Spm was conjugated, via TGase, mainly to 22–30 kDa proteins. Long-term experiments over 5 days showed a general decrease in all three parameters with or without Spm. When leaves remained on the plants, Spm-sprayed leaves showed an increase in free Spm 1 h after spraying, mainly in the young leaves, whereas over longer periods (15 days) there was an increase in perchloric acid-soluble and -insoluble Spm metabolites. In senescing leaves, Spm prevented degradation of chlorophyll b and some proteins, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, but also those enriched in PSI, whose light-harvesting complexes (LHC) sub-fractions contained TGase. Spm was conjugated by TGase mainly to LHCII, more markedly in the light. Immunodetection of TGase revealed multiple proteins in young leaves, possibly representing different TGase isoforms when TGase activity was high, whereas in already senescent leaves, when its activity decreased, one high-molecular-mass band was found, possibly because of enzyme polymerisation. Spm thus protected senescing Lactuca leaves from the decay of their chloroplast photosystem complexes. The senescence-delaying effects of Spm could be mediated by TGase, as TGase was re-activated to the level in young leaves following Spm treatment.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.