Enzymes Of Chlorophyll Biosynthesis Research Articles

Chemical Composition, Repellent, and Phytotoxic Potentials of the Fractionated Resin Essential Oil from Araucaria heterophylla Growing in Tunisia.

The resin essential oil (REO) of the Tunisian Araucaria heterophylla trunk bark was investigated for its chemical composition. Then, it was evaluated for its insecticidal and allelopathic activities. The REO was obtained by hydrodistillation for 9 h (yield of 4.2 % w/w). Moreover, fractional hydrodistillation was carried out at 3-hour intervals, resulting in 3 fractions (R1-R3), to facilitate chemical identification and localization of the aforementioned biological activities. GC/MS analysis of the obtained samples allowed the identification of 25 compounds, representing between 91.2 and 96.3 % of their total constituents, which consisted predominantly of sesquiterpene hydrocarbons, oxygenated sesquiterpenes and diterpene hydrocarbons. α-Copaene (10.8 %), γ-muurolene (5.8 %), α-copaen-11-ol (7.8 %), spathulenol (10.5 %), 15-copaenol (8.2 %), ylangenal (10.3 %), dehydrosaussurea lactone (7.7 %), and sandaracopimaradiene (11.4 %) were identified as major compounds. The second part aimed to assess the impact of the A. heterophylla EO and its three fractions for their insecticidal and repellent activity against Tribolium castaneum (Herbst), a stored grain pest, of which a strong repellent activity was noted. In addition, the studied samples showed high phytotoxic effects against Lactuca sativa. The third fraction (R3) performed a total inhibitory potential on seed germination and seedling growth of the target plant. Furthermore, alongside this discovery, an estimation was conducted through molecular docking analysis. Wherein the main compounds of the studied samples were docked into the active pocket of protoporphyrinogen IX oxidase (PDB: 1SEZ), a key enzyme in chlorophyll biosynthesis. Thus, it is recommended to use the REO of A. heterophylla as a natural herbicide.

The Crucial Role of SlGSNOR in Regulating Postharvest Tomato Fruit Ripening.

S-nitrosoglutathione reductase (GSNOR) is a well-known regulator in controlling protein S-nitrosylation modification and nitric oxide (NO) homeostasis. Here, a GSNOR inhibitor N6022 and SlGSNOR silencing were applied to investigate the roles of SlGSNOR in tomato fruit postharvest ripening. We found that the application of N6022 and S-nitrosoglutathione (GSNO, a NO donor), and SlGSNOR silencing delayed the transition of fruit skin color by improving total chlorophyll level by 88.57%, 44.78%, and 91.03%, respectively. Meanwhile, total carotenoid and lycopene contents were reduced by these treatments. Concurrently, the activity of chlorophyll biosynthesis enzymes and the expression of related genes were upregulated, and the transcript abundances of total carotenoid bioproduction genes were downregulated, by N6022 and GSNO treatments and SlGSNOR silencing. In addition, fruit softening was postponed by N6022, GSNO, and SlGSNOR silencing, through delaying the decrease of firmness and declining cell wall composition; structure-related enzyme activity; and gene expression levels. Furthermore, N6022, GSNO, and SlGSNOR silencing enhanced the accumulation of titratable acid; ascorbic acid; total phenol; and total flavonoid, but repressed the content of soluble sugar and soluble protein accompanied with the expression pattern changes of nutrition-related genes. In addition, the endogenous NO contents were elevated by 197.55%; 404.59%; and 713.46%, and the endogenous SNOs contents were enhanced by 74.65%; 93.49%; and 94.85%; by N6022 and GSNO treatments and SlGSNOR silencing, respectively. Altogether, these results indicate that SlGSNOR positively promotes tomato postharvest fruit ripening, which may be largely on account of its negative roles in the endogenous NO level.

NTRC and TRX-f Coordinately Affect the Levels of Enzymes of Chlorophyll Biosynthesis in a Light-Dependent Manner.

Redox regulation of plastid gene expression and different metabolic pathways promotes many activities of redox-sensitive proteins. We address the question of how the plastid redox state and the contributing reducing enzymes control the enzymes of tetrapyrrole biosynthesis (TBS). In higher plants, this metabolic pathway serves to produce chlorophyll and heme, among other essential end products. Because of the strictly light-dependent synthesis of chlorophyll, tight control of TBS requires a diurnal balanced supply of the precursor 5-aminolevulinic acid (ALA) to prevent the accumulation of photoreactive metabolic intermediates in darkness. We report on some TBS enzymes that accumulate in a light intensity-dependent manner, and their contents decrease under oxidizing conditions of darkness, low light conditions, or in the absence of NADPH-dependent thioredoxin reductase (NTRC) and thioredoxin f1 (TRX-f1). Analysis of single and double trxf1 and ntrc mutants revealed a decreased content of the early TBS enzymes glutamyl-tRNA reductase (GluTR) and 5-aminolevulinic acid dehydratase (ALAD) instead of an exclusive decrease in enzyme activity. This effect was dependent on light conditions and strongly attenuated after transfer to high light intensities. Thus, it is suggested that a deficiency of plastid-localized thiol-redox transmitters leads to enhanced degradation of TBS enzymes rather than being directly caused by lower catalytic activity. The effects of the proteolytic activity of the Clp protease on TBS enzymes were studied by using Clp subunit-deficient mutants. The simultaneous lack of TRX and Clp activities in double mutants confirms the Clp-induced degradation of some TBS proteins in the absence of reductive activity of TRXs. In addition, we verified previous observations that decreased chlorophyll and heme levels in ntrc could be reverted to WT levels in the ntrc/Δ2cp triple mutant. The decreased synthesis of 5-aminolevulinic acid and porphobilinogen in ntrc was completely restored in ntrc/Δ2cp and correlated with WT-like levels of GluTR, ALAD, and other TBS proteins.

Chloroplast SRP43 and SRP54 independently promote thermostability and membrane binding of light-dependent protochlorophyllide oxidoreductases.

Protochlorophyllide oxidoreductase (POR), which converts protochlorophyllide into chlorophyllide, is the only light-dependent enzyme in chlorophyll biosynthesis. While its catalytic reaction and importance for chloroplast development are well understood, little is known about the post-translational control of PORs. Here, we show that cpSRP43 and cpSRP54, two components of the chloroplast signal recognition particle pathway, play distinct roles in optimizing the function of PORB, the predominant POR isoform in Arabidopsis. The chaperone cpSRP43 stabilizes the enzyme and provides appropriate amounts of PORB during leaf greening and heat shock, whereas cpSRP54 enhances its binding to the thylakoid membrane, thereby ensuring adequate levels of metabolic flux in late chlorophyll biosynthesis. Furthermore, cpSRP43 and the DnaJ-like protein CHAPERONE-LIKE PROTEIN of POR1 concurrently act to stabilize PORB. Overall, these findings enhance our understanding of the coordinating role of cpSPR43 and cpSRP54 in the post-translational control of chlorophyll synthesis and assembly of photosynthetic chlorophyll-binding proteins.

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 420ps. 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.

Mapping of a Pale Green Mutant Gene and Its Functional Verification by Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaves are the main organ for photosynthesis, and variations in leaf color affect photosynthesis and plant biomass formation. We created two similar whole-plant pale green mutants (pem1 and pem2) from the double haploid (DH) Chinese cabbage line “FT” through ethyl methanesulfonate (EMS) mutagenesis of seeds. Photosynthetic pigment contents and net photosynthetic rates were significantly lower in the mutants than in the wild-type “FT,” and the chloroplast thylakoid endomembrane system was poor. Genetic analysis showed that the mutated phenotypes of pem1 and pem2 were caused by a single nuclear gene. Allelism tests showed that pem1 and pem2 were alleles. We mapped Brpem1 to a 64.25 kb region on chromosome A10, using BSR-Seq and map-based cloning of 979 F2 recessive individuals. Whole-genome re-sequencing revealed a single nucleotide polymorphism (SNP) transition from guanine to adenosine on BraA10g021490.3C in pem1, causing an amino acid shift from glycine to glutamic acid (G to E); in addition, BraA10g021490.3C in pem2 was found to have a single nucleotide substitution from guanine to adenosine, causing an amino acid change from E to lysine (K). BraA10g021490.3C is a homolog of the Arabidopsisdivinyl chlorophyllide a 8-vinyl-reductase (DVR) gene that encodes 3,8-divinyl protochlorophyllide a 8-vinyl reductase, which is a key enzyme in chlorophyll biosynthesis. Enzyme activity assay and chlorophyll composition analysis demonstrated that impaired DVR had partial loss of function. These results provide a basis to understand chlorophyll metabolism and explore the mechanism of a pale green phenotype in Chinese cabbage.

Chaperone-like protein DAY plays critical roles in photomorphogenesis

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARKANDYELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

Elucidation of ligand binding and dimerization of NADPH:protochlorophyllide (Pchlide) oxidoreductase from pea (Pisum sativum L.) by structural analysis and simulations.

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. It is one of few known photoenzymes, which catalyzes the light-activated trans-reduction of the C17-C18 double bond of Pchlide's porphyrin ring. Due to the light requirement, dark-grown angiosperms cannot synthesize chlorophyll. No crystal structure of POR is available, so to improve understanding of the protein's three-dimensional structure, its dimerization, and binding of ligands (both the cofactor NADPH and substrate Pchlide), we computationally investigated the sequence and structural relationships among homologous proteins identified through database searches. The results indicate that α4 and α7 helices of monomers form the interface of POR dimers. On the basis of conserved residues, we predicted 11 functionally important amino acids that play important roles in POR binding to NADPH. Structural comparison of available crystal structures revealed that they participate in formation of binding pockets that accommodate the Pchlide ligand, and that five atoms of the closed tetrapyrrole are involved in non-bonding interactions. However, we detected no clear pattern in the physico-chemical characteristics of the amino acids they interact with. Thus, we hypothesize that interactions of these atoms in the Pchlide porphyrin ring are important to hold the ligand within the POR binding site. Analysis of Pchlide binding in POR by molecular docking and PELE simulations revealed that the orientation of the nicotinamide group is important for Pchlide binding. These findings highlight the complexity of interactions of porphyrin-containing ligands with proteins, and we suggest that fit-inducing processes play important roles in POR-Pchlide interactions.

Xanthophyll carotenoids stabilise the association of cyanobacterial chlorophyll synthase with the LHC-like protein HliD

Chlorophyll synthase (ChlG) catalyses a terminal reaction in the chlorophyll biosynthesis pathway, attachment of phytol or geranylgeraniol to the C17 propionate of chlorophyllide. Cyanobacterial ChlG forms a stable complex with high light-inducible protein D (HliD), a small single-helix protein homologous to the third transmembrane helix of plant light-harvesting complexes (LHCs). The ChlG-HliD assembly binds chlorophyll, β-carotene, zeaxanthin and myxoxanthophyll and associates with the YidC insertase, most likely to facilitate incorporation of chlorophyll into translated photosystem apoproteins. HliD independently coordinates chlorophyll and β-carotene but the role of the xanthophylls, which appear to be exclusive to the core ChlG-HliD assembly, is unclear. Here we generated mutants of Synechocystis sp. PCC 6803 lacking specific combinations of carotenoids or HliD in a background with FLAG- or His-tagged ChlG. Immunoprecipitation experiments and analysis of isolated membranes demonstrate that the absence of zeaxanthin and myxoxanthophyll significantly weakens the interaction between HliD and ChlG. ChlG alone does not bind carotenoids and accumulation of the chlorophyllide substrate in the absence of xanthophylls indicates that activity/stability of the 'naked' enzyme is perturbed. In contrast, the interaction of HliD with a second partner, the photosystem II assembly factor Ycf39, is preserved in the absence of xanthophylls. We propose that xanthophylls are required for the stable association of ChlG and HliD, acting as a 'molecular glue' at the lateral transmembrane interface between these proteins; roles for zeaxanthin and myxoxanthophyll in ChlG-HliD complexation are discussed, as well as the possible presence of similar complexes between LHC-like proteins and chlorophyll biosynthesis enzymes in plants.

A point mutation in the photosystem I P700 chlorophyll a apoprotein A1 gene confers variegation in Helianthus annuus L.

Even a point mutation in the psaA gene mediates chlorophyll deficiency. The role of the plastid signal may perform the redox state of the compounds on the acceptor-side of PSI. Two extranuclear variegated mutants of sunflower, Var1 and Var33, were investigated. The yellow sectors of both mutants were characterized by an extremely low chlorophyll and carotenoid content, as well as poorly developed, unstacked thylakoid membranes. A full-genome sequencing of the cpDNA revealed mutations in the psaA gene in both Var1 and Var33. The cpDNA from the yellow sectors of Var1 differs from those in the wild type by only a single, non-synonymous substitution (Gly734Glu) in the psaA gene, which encodes a subunit of photosystem (PS) I. In the cpDNA from the yellow sectors of Var33, the single-nucleotide insertion in the psaA gene was revealed, leading to frameshift at the 580 amino acid position. Analysis of the photosynthetic electron transport demonstrated an inhibition of the PSI and PSII activities in the yellow tissues of the mutant plants. It has been suggested that mutations in the psaA gene of both Var1 and Var33 led to the disruption of PSI. Due to the non-functional PSI, photosynthetic electron transport is blocked, which, in turn, leads to photodamage of PSII. These data are confirmed by immunoblotting analysis, which showed a significant reduction in PsbA in the yellow leaf sectors, but not PsaA. The expression of chloroplast and nuclear genes encoding the PSI subunits (psaA, psaB, and PSAN), the PSII subunits (psbA, psbB, and PSBW), the antenna proteins (LHCA1, LHCB1, and LHCB4), the ribulose 1.5-bisphosphate carboxylase subunits (rbcL and RbcS), and enzymes of chlorophyll biosynthesis were down-regulated in the yellow leaf tissue. The extremely reduced transcriptional activity of the two protochlorophyllide oxidoreductase (POR) genes involved in chlorophyll biosynthesis is noteworthy. The disruption of NADPH synthesis, due to the non-functional PSI, probably led to a significant reduction in NADPH-protochlorophyllide oxidoreductase in the yellow sectors of Var1 and Var33. A dramatic decrease in chlorophyllide was shown in the yellow sectors. A reduction in NADPH-protochlorophyllide oxidoreductase, along with photodegradation, has been suggested as a result of chlorophyll deficiency.

Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae

Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 ( Wang et al., 2020 ). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro enzymatic assay of magnesium chelatase ( Wang et al., 2020 ). Currently, the crystallization studies of the BCM1 are underway. This protocol could be adapted to purify BCM1-like transmembrane proteins from eukaryotic organisms for enzymatic and structural studies.

Influence of Blue-Light Signaling Components on the Regulation of Cytokinin-Dependent Arabidopsis thaliana Seedlings’ Greening

De-etiolation of plant seedlings is controlled by a complex system of light and hormonal signaling. This process is accompanied by chloroplast development and initiation of chlorophyll biosynthesis leading to cotyledon greening. In this study, peculiaritis of regulation of expression of the key genes encoding enzymes of chlorophyll biosynthesis in wild type plants and mutants of perception and transduction of blue light in Arabidopsis thaliana (L.) Heynh. during cytokinin (CK)-dependent greening of seedlings were investigated. Inactivation of blue-light signaling components (??RY1, CRY2 and HY5) led to retarded chlorophyll accumulation and decrease of the expression of the key genes responsible for its biosynthesis. Exogenous application of cytokinin accelerated de-etiolation of wild type and knockout-mutants cry1cry2 and hy5 seedlings that was manifested itself in elevating chlorophyll a fluorescence and higher level of transcripts of a number of genes involved in chlorophyll biosynthesis. Taken together the results may provide evidence for the significant contribution of blue-light signaling to the greening regulation at early steps of de-etiolation of seedlings and involvement of HY5 protein in realization of the positive effect of CK at early steps of photomorphogenesis.

Overexpression of CONSTANS-like 16 enhances chlorophyll accumulation in petunia corollas

We have previously found that a gene closely related to Arabidopsis CONSTANS-like 16 (COL16) was coordinately expressed with chlorophyll content in chrysanthemum petals and leaves. Here, to elucidate whether COL16 is involved in the regulation of chlorophyll biosynthesis and accumulation, we analyzed the function of COL16 in petunia (Petunia hybrida). We identified three petunia COL16 homologs: PhCOL16a, PhCOL16b, and PhCOL16c. Expression patterns of all three homologs were associated with chlorophyll content, with lower levels in white corollas than in pale green corollas, and relatively high levels in leaves. The result suggests that PhCOL16 homologs are involved in chlorophyll accumulation. We introduced a PhCOL16a overexpression construct into petunia. The transgenic plants had pale green corollas with a higher chlorophyll content than wild-type plants. Expression of genes encoding key enzymes of chlorophyll biosynthesis was significantly higher in the transgenic plants than in the wild-type plants. The results indicate that PhCOL16 positively regulates chlorophyll biosynthesis.

Plant and algal chlorophyll synthases function in Synechocystis and interact with the YidC/Alb3 membrane insertase.

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light‐inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co‐ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG‐YidC/Alb3 complex may be evolutionarily conserved in algae and plants.

Redox-control of chlorophyll biosynthesis mainly depends on thioredoxins.

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the palegreen phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes.

N-terminus plus linker domain of Mg-chelatase D subunit is essential for Mg-chelatase activity in Oryza sativa

Mg chelatase, a key enzyme in chlorophyll biosynthesis, is comprised of I, D and H subunits. Among these subunits, the D subunit was regarded to mediate protein interactions due to its unique protein domains. However, the functional roles of the different domains of the D subunit in vivo remain unclear. In this study, we dissected the rice (Oryza sativa) D subunit (OsCHLD) into three peptide fragments: the putative chloroplast transit peptide (TP, Met1 to Arg45), the N-terminus plus linker domain (OsCHLDN + L, Ala46 to Leu485) and the C-terminus (OsCHLDC, Ile486 to Ser754), to explore the roles of these fragments. The results of the yeast two-hybrid assay and the in vitro reconstitution of the Mg-chelatase activity showed that only OsCHLDN + L interacted with the I and H subunits and maintained most of the Mg-chelatase activity in vitro. Furthermore, artificial TP-OsCHLDN + L and TP-OsCHLDC were overexpressed in rice. Interestingly, an incomplete co-suppression had occurred in both of the overexpressed (OsCHLDN + L-ox and OsCHLDC-ox) plants, resulting in a significantly downregulated expression of endogenous OsCHLD. Therefore, these transgenic plants had adequate OsCHLDN + L and OsCHLDC instead of endogenous OsCHLD, providing ideal models to study the function of different domains of the D subunit in vivo. The OsCHLDN + L-ox plants showed an identical phenotype to that of the wild type, while the OsCHLDC-ox plants demonstrated a yellowish phenotype that resembled the D subunit mutants. These results indicated that only OsCHLDN + L could complement the function of endogenous OsCHLD, providing direct evidence that OsCHLDN + L is essential for Mg-chelatase activity in vivo.

AhGLK1 affects chlorophyll biosynthesis and photosynthesis in peanut leaves during recovery from drought

Peanut is an important edible oil crop plant whose quality and yield are greatly affected by drought. The process and molecular mechanisms of recovery from drought are also critical to its productivity, but are currently poorly characterized. Here, we investigate the involvement of peanut AhGLK1 in recovery from drought, and in particular its relationship with AhPORA, which encodes a key enzyme in chlorophyll biosynthesis. We found that chlorophyll content, chlorophyll fluorescence, AhPORA protein level and genes related to chlorophyll biosynthesis and photosynthesis declined markedly under drought conditions, but all increased during recovery. Consistent with this, AhGLK1 expression decreased during water stress and increased when the stress was removed. When AhGLK1 was transformed into Arabidopsis glk1glk2 mutant, it increased the survival rate of the mutant during recovery from drought and fully rescued the mutant’s pale-green phenotype. In addition, chlorophyll content and fluorescence, and the expression of genes related to chlorophyll biosynthesis and photosynthesis, were all increased. Bioinformatics analysis and experimental evidence suggested that AhGLK1 augments the expression of AhPORA by binding to its promoter. Our findings confirm that AhGLK1 plays a role as a transcription factor that upregulates expression of AhPORA during post-drought recovery, thereby stimulating chlorophyll biosynthesis and photosynthesis.

Mutation in Mg-Protoporphyrin IX Monomethyl Ester Cyclase Decreases Photosynthesis Capacity in Rice.

In photosynthesis, the pigments chlorophyll a/b absorb light energy to convert to chemical energy in chloroplasts. Though most enzymes of chlorophyll biosynthesis from glutamyl-tRNA to chlorophyll a/b have been identified, the exact composition and regulation of the multimeric enzyme Mg-protoporphyrin IX monomethyl ester cyclase (MPEC) is largely unknown. In this study, we isolated a rice pale-green leaf mutant m167 with yellow-green leaf phenotype across the whole lifespan. Chlorophyll content decreases 43–51% and the granal stacks of chloroplasts becomes thinner in m167. Chlorophyll fluorescence parameters, including Fv/Fm (the maximum quantum efficiency of PSII) and quantum yield of PSII (Y(II)), were lower in m167 than those in wild type plants (WT), and photosynthesis rate decreases 40% in leaves of m167 mutant compared with WT plants, which lead to yield reduction in m167. Genetic analysis revealed that yellow-green leaf phenotype of m167 is controlled by a single recessive genetic locus. By positional cloning, a single mutated locus, G286A (Alanine 96 to Threonine in protein), was found in the coding sequence of LOC_Os01g17170 (Rice Copper Response Defect 1, OsCRD1), encoding a putative subunit of MPEC. Expression profile analysis demonstrated that OsCRD1 is mainly expressed in green tissues of rice. Sequence alignment analysis of CRD1 indicated that Alanine 96 is very conserved in all green plants and photosynthetic bacteria. OsCRD1 protein mainly locates in chloroplast and the point mutation A96T in OsCRD1 does not change its location. Therefore, Alanine96 of OsCRD1 might be fundamental for MPEC activity, mutation of which leads to deficiency in chlorophyll biosynthesis and chloroplast development and decreases photosynthetic capacity in rice.

Identification of genes associated with chlorophyll accumulation in flower petals.

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.