Tocopherol and phylloquinone biosynthesis in chloroplasts requires the phytol kinase VITAMIN E PATHWAY GENE5 (VTE5) and the farnesol kinase (FOLK).

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK.

Abscisic acid triggers vitamin E accumulation by transient transcript activation of VTE5 and VTE6 in sweet cherry fruits.

Tocopherols are lipophilic antioxidants known as vitamin E and synthesized from the condensation of two metabolic pathways leading to the formation of homogentisate and phytyl diphosphate. While homogentisate is derived from tyrosine metabolism, phytyl diphosphate may be formed from geranylgeranyl diphosphate or phytol recycling from chlorophyll degradation. Here, we hypothesized that abscisic acid (ABA) could induce tocopherol biosynthesis in sweet cherries by modifying the expression of genes involved in vitamin E biosynthesis, including those from the phytol recycling pathway. Hence, the expression of key tocopherol biosynthesis genes was determined together with vitamin E and chlorophyll contents during the natural development of sweet cherries on the tree. Moreover, the effects of exogenously applied ABA on the expression of key tocopherol biosynthesis genes were also investigated during on-tree fruit development, and tocopherols and chlorophylls contents were analyzed. Results showed that the expression of tocopherol biosynthesis genes, including VTE5, VTE6, HPPD and HPT showed contrasting patterns of variation, but in all cases, increased by 2- and 3-fold over time during fruit de-greening. This was not the case for GGDR and VTE4, the first showing constitutive expression during fruit development and the second with marked down-regulation at ripening onset. Furthermore, exogenous ABA stimulated the production of both α- and γ-tocopherols by 60% and 30%, respectively, promoted chlorophyll degradation and significantly enhanced VTE5 and VTE6 expression, and also that of HPPD and VTE4, altogether increasing total tocopherol accumulation. In conclusion, ABA increases promote the transcription of phytol recycling enzymes, which may contribute to vitamin E biosynthesis during fruit development in stone fruits like sweet cherries.

Genome-wide association identifies a missing hydrolase for tocopherol synthesis in plants.

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

2-Hydroxy-phytanoyl-CoA lyase (AtHPCL) is involved in phytol metabolism in Arabidopsis.

During chlorophyll degradation, large amounts of the isoprenoid alcohol phytol are released. The pathway of phytol catabolism has been studied in humans, because chlorophyll is part of the human diet, but little is known for plants. In humans, phytanoyl-CoA derived from phytol is degraded via α-oxidation by phytanoyl-CoA hydroxylase (PAHX) and 2-hydroxy-phytanoyl-CoA lyase (HPCL). Arabidopsis contains two sequences homologous to the human proteins AtPAHX and AtHPCL. Insertional mutants of Arabidopsis (pahx, hpcl) were grown under N deprivation to stimulate chlorophyll breakdown or supplemented with phytol to increase the endogenous amount of phytol. During N deprivation, chlorophyll, phytol, phytenal, upstream metabolites of phytol breakdown, and tocopherol and fatty acid phytyl esters, alternative phytol-derived lipids, accumulated in pahx and hpcl mutants, in line with the scenario that the mutations interfere with phytol degradation. AtHPCL was localized to the peroxisomes. Expression analysis of the AtHPCL sequence in the yeast Δpxp1 or Δmpo1 mutants followed by supplementation with 2-hydroxy-palmitic acid and enzyme assays of peroxisomal proteins from Col-0 and hpcl plants with 2-hydroxy-stearoyl-CoA revealed that AtHPCL harbors 2-hydroxy-acyl-CoA lyase activity. The α-dioxygenases αDOX1 and αDOX2 are involved in α-oxidation of fatty acids and could be involved in an alternative pathway of phytol degradation. However, phytol-related lipids in the αdox1, αdox2, or αdox1 αdox2 mutants were not altered compared with Col-0, indicating that αDOX1 and αDOX2 are not involved in phytol degradation. These results demonstrate that phytol degradation in Arabidopsis involves α-oxidation by AtPAHX and AtHPCL, but that it is independent of αDOX1/αDOX2.

Phytol recycling: essential, yet not limiting for tomato fruit tocopherol accumulation under normal growing conditions.

Tocopherols are potent membrane-bound antioxidant molecules that are paramount for plant physiology and also important for human health. In the past years, chlorophyll catabolism was identified as the primary source of phytyl diphosphate for tocopherol synthesis by the action of two enzymes, PHYTOL KINASE (VTE5) and PHYTHYL PHOSPHATE KINASE (VTE6) that are able to recycle the chlorophyll-derived phytol. While VTE5 and VTE6 were proven essential for tocopherol metabolism in tomato fruits, it remains unknown whether they are rate-limiting steps in this pathway. To address this question, transgenic tomato plants expressing AtVTE5 and AtVTE6 in a fruit-specific manner were generated. Although ripe transgenic fruits exhibited higher amounts of tocopherol, phytol recycling revealed a more intimate association with chlorophyll than with tocopherol content. Interestingly, protein-protein interactions assays showed that VTE5 and VTE6 are complexed, channeling free phytol and phytyl-P, thus mitigating their cytotoxic nature. Moreover, the analysis of tocopherol accumulation dynamics in roots, a chlorophyll-devoid organ, revealed VTE5-dependent tocopherol accumulation, hinting at the occurrence of shoot-to-root phytol trafficking. Collectively, these results demonstrate that phytol recycling is essential for tocopherol biosynthesis, even in chlorophyll-devoid organs, yet it is not the rate-limiting step for this pathway under normal growth conditions.

Triacylglycerol and phytyl ester synthesis in Synechocystis sp. PCC6803.

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

The tail of chlorophyll: Fates for phytol.

Understanding the pathways involved in chlorophyll breakdown provides a molecular map to the color changes observed in plant life on a global scale each fall. Surprisingly, little is known about the fate of phytol, chlorophyll's 20-carbon branched-chain tail, during this process. A recent study from Gutbrod et al. provides evidence using physiological, genetic, and exquisitely sensitive analytical approaches that phytenal is an intermediate in plant phytol catabolism. These insights and techniques open the door to further investigation of this complicated metabolic system, with implications for plant health and agriculture.

Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal.

Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable. We found that phytenal accumulates during chlorotic stresses, for example, salt stress, dark-induced senescence, and nitrogen deprivation. The increase in the phytenal content is mediated at least in part independently of enzyme activities, and it is independent of light. Characterization of phytenal accumulation in the pao1 mutant affected in chlorophyll degradation revealed that phytenal is an authentic phytol metabolite derived from chlorophyll breakdown. The increase in phytenal was even stronger in mutants affected in the production of other phytol metabolites including vte5-2 (tocopherol deficient) and pes1 pes2 (fatty acid phytyl ester deficient). Therefore, phytenal accumulation is controlled by competing, alternative pathways of phosphorylation (leading to tocopherol production) or esterification (fatty acid phytyl ester production). As a consequence, the content of phytenal is maintained at low levels, presumably to minimize its toxic effects caused by its highly reactive aldehyde group that can form covalent bonds with and inactivate the amino groups of proteins.

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize.

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8-bp insertion/deletion (InDel058) in the 5' untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA-sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR-based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.

Phytol, a Chlorophyll Component, Produces Antihyperalgesic, Anti-inflammatory, and Antiarthritic Effects: Possible NFκB Pathway Involvement and Reduced Levels of the Proinflammatory Cytokines TNF-α and IL-6.

Phytol is a diterpene constituent of chlorophyll and has been shown to have several pharmacological properties, particularly in relation to the management of painful inflammatory diseases. Arthritis is one of the most common of these inflammatory diseases, mainly affecting the synovial membrane, cartilage, and bone in joints. Proinflammatory cytokines, such as TNF-α and IL-6, and the NFκB signaling pathway play a pivotal role in arthritis. However, as the mechanisms of action of phytol and its ability to reduce the levels of these cytokines are poorly understood, we decided to investigate its pharmacological effects using a mouse model of complete Freund's adjuvant (CFA)-induced arthritis. Our results showed that phytol was able to inhibit joint swelling and hyperalgesia throughout the whole treatment period. Moreover, phytol reduced myeloperoxidase (MPO) activity and proinflammatory cytokine release in synovial fluid and decreased IL-6 production as well as the COX-2 immunocontent in the spinal cord. It also downregulated the p38MAPK and NFκB signaling pathways. Therefore, our findings demonstrated that phytol can be an innovative antiarthritic agent due to its capacity to attenuate inflammatory reactions in joints and the spinal cord, mainly through the modulation of mediators that are key to the establishment of arthritic pain.

Transcription factor RD26 is a key regulator of metabolic reprogramming during dark-induced senescence.

Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 overexpressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced γ-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono- and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis.

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.

Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice.

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.

Essential role for phytol kinase and tocopherol in tolerance to combined light and temperature stress in tomato.

In a changing environment, plants need to cope with the impact of rising temperatures together with high light intensity. Here, we used lipidomics in the tomato model system to identify lipophilic molecules that enhance tolerance to combined high-temperature and high-light stress. Among several hundred metabolites, the two most strongly up-regulated compounds were α-tocopherol and plastoquinone/plastoquinol. Both are well-known lipid antioxidants and contribute to the protection of photosystem II (PSII) against photodamage under environmental stress. To address the protective function of tocopherol, an RNAi line (vte5) with decreased expression of VTE5 and reduced levels of α-tocopherol was selected. VTE5 encodes phytol kinase, which acts in the biosynthetic pathway of tocopherols. vte5 suffered strong photoinhibition and photobleaching when exposed to combined high-light and high-temperature stress, but neither stress alone produced a visible phenotype. As vte5 had plastoquinone levels similar to those of the wild type under combined stress, the strong phenotype could be attributed to the lack of α-tocopherol. These findings suggest that VTE5 protects against combined high-light and high-temperature stress and does so by supporting α-tocopherol production.

Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in Arabidopsis.

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide-resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide-resistant respiration was also accompanied by the increase of plastid NADPH/NADP(+) ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin-Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX-derived plastidial NADPH/NADP(+) ratio change. Further, our results thus revealed a molecular mechanism of chloroplast-mitochondria interactions.

Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner.

Tocopherol, a compound with vitamin E (VTE) activity, is a conserved constituent of the plastidial antioxidant network in photosynthetic organisms. The synthesis of tocopherol involves the condensation of an aromatic head group with an isoprenoid prenyl side chain. The latter, phytyl diphosphate, can be derived from chlorophyll phytol tail recycling, which depends on phytol kinase (VTE5) activity. How plants co-ordinate isoprenoid precursor distribution for supplying biosynthesis of tocopherol and other prenyllipids in different organs is poorly understood. Here, Solanum lycopersicum plants impaired in the expression of two VTE5-like genes identified by phylogenetic analyses, named SlVTE5 and SlFOLK, were characterized. Our data show that while SlFOLK does not affect tocopherol content, the production of this metabolite is >80% dependent on SlVTE5 in tomato, in both leaves and fruits. VTE5 deficiency greatly impacted lipid metabolism, including prenylquinones, carotenoids, and fatty acid phytyl esters. However, the prenyllipid profile greatly differed between source and sink organs, revealing organ-specific metabolic adjustments in tomato. Additionally, VTE5-deficient plants displayed starch accumulation and lower CO2 assimilation in leaves associated with mild yield penalty. Taken together, our results provide valuable insights into the distinct regulation of isoprenoid metabolism in leaves and fruits and also expose the interaction between lipid and carbon metabolism, which results in carbohydrate export blockage in the VTE5-deficient plants, affecting tomato fruit quality.

Functional analysis of light-harvesting-like protein 3 (LIL3) and its light-harvesting chlorophyll-binding motif in Arabidopsis.

The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25-30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.

Different mechanisms are responsible for chlorophyll dephytylation during fruit ripening and leaf senescence in tomato.

Chlorophyll breakdown occurs in different green plant tissues (e.g. during leaf senescence and in ripening fruits). For different plant species, the PHEOPHORBIDE A OXYGENASE (PAO)/phyllobilin pathway has been described to be the major chlorophyll catabolic pathway. In this pathway, pheophorbide (i.e. magnesium- and phytol-free chlorophyll) occurs as a core intermediate. Most of the enzymes involved in the PAO/phyllobilin pathway are known; however, the mechanism of dephytylation remains uncertain. During Arabidopsis (Arabidopsis thaliana) leaf senescence, phytol hydrolysis is catalyzed by PHEOPHYTINASE (PPH), which is specific for pheophytin (i.e. magnesium-free chlorophyll). By contrast, in fruits of different Citrus spp., chlorophyllase, hydrolyzing phytol from chlorophyll, was shown to be active. Here, we enlighten the process of chlorophyll breakdown in tomato (Solanum lycopersicum), both in leaves and fruits. We demonstrate the activity of the PAO/phyllobilin pathway and identify tomato PPH (SlPPH), which, like its Arabidopsis ortholog, was specifically active on pheophytin. SlPPH localized to chloroplasts and was transcriptionally up-regulated during leaf senescence and fruit ripening. SlPPH-silencing tomato lines were impaired in chlorophyll breakdown and accumulated pheophytin during leaf senescence. However, although pheophytin transiently accumulated in ripening fruits of SlPPH-silencing lines, ultimately these fruits were able to degrade chlorophyll like the wild type. We conclude that PPH is the core phytol-hydrolytic enzyme during leaf senescence in different plant species; however, fruit ripening involves other hydrolases, which are active in parallel to PPH or are the core hydrolases in fruits. These hydrolases remain unidentified, and we discuss the question of whether chlorophyllases might be involved.

Chlorophyll degradation: the tocopherol biosynthesis-related phytol hydrolase in Arabidopsis seeds is still missing.

Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn't enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis.

Purification and immobilization of the recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as potential biocatalyst for the production of chlorophyllide and phytol.

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.