Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Chemical Profile of Cyperus laevigatus and Its Protective Effects against Thioacetamide-Induced Hepatorenal Toxicity in Rats

Cyperus species represent a group of cosmopolitan plants used in folk medicine to treat several diseases. In the current study, the phytochemical profile of Cyperus laevigatus ethanolic extract (CLEE) was assessed using UPLC-QTOF–MS/MS. The protective effect of CLEE at 50 and 100 mg /kg body weight (b.w.) was evaluated on hepatorenal injuries induced by thioacetamide (100 mg/kg) via investigation of the extract’s effects on oxidative stress, inflammatory markers and histopathological changes in the liver and kidney. UPLC-QTOF–MS/MS analysis of CLEE resulted in the identification of 94 compounds, including organic and phenolic acids, flavones, aurones, and fatty acids. CLEE improved the antioxidant status in the liver and kidney, as manifested by enhancement of reduced glutathione (GSH) and coenzyme Q10 (CoQ10), in addition to the reduction in malondialdehyde (MDA), nitric oxide (NO), and 8-hydroxy-2′-deoxyguanosine (8OHdG). Moreover, CLEE positively affected oxidative stress parameters in plasma and thwarted the depletion of hepatorenal ATP content by thioacetamide (TAA). Furthermore, treatment of rats with CLEE alleviated the significant increase in plasma liver enzymes, kidney function parameters, and inflammatory markers. The protective effect of CLEE was confirmed by a histopathological study of the liver and kidney. Our results proposed that CLEE may reduce TAA-hepatorenal toxicity via its antioxidant and anti-inflammatory properties suppressing oxidative stress

Viewing oxidative stress through the lens of oxidative signalling rather than damage

Concepts of the roles of reactive oxygen species (ROS) in plants and animals have shifted in recent years from focusing on oxidative damage effects to the current view of ROS as universal signalling metabolites. Rather than having two opposing activities, i.e. damage and signalling, the emerging concept is that all types of oxidative modification/damage are involved in signalling, not least in the induction of repair processes. Examining the multifaceted roles of ROS as crucial cellular signals, we highlight as an example the loss of PSII function called photoinhibition, where photo-protection has classically been conflated with oxidative damage

Ascorbate-mediated regulation of growth, photoprotection, and photoinhibition in Arabidopsis thaliana.

The requirements for ascorbate for growth and photosynthesis were assessed under low (LL; 250 µmol m-2 s-1) or high (HL; 1600 µmol m-2 s-1) irradiance in wild-type Arabidopsis thaliana and two ascorbate synthesis mutants (vtc2-1 and vtc2-4) that have 30% wild-type ascorbate levels. The low ascorbate mutants had the same numbers of leaves but lower rosette area and biomass than the wild type under LL. Wild-type plants experiencing HL had higher leaf ascorbate, anthocyanin, and xanthophyll pigments than under LL. In contrast, leaf ascorbate levels were not increased under HL in the mutant lines. While the degree of oxidation measured using an in vivo redox reporter in the nuclei and cytosol of the leaf epidermal and stomatal cells was similar under both irradiances in all lines, anthocyanin levels were significantly lower in the low ascorbate mutants than in the wild type under HL. Differences in the photosynthetic responses of vtc2-1 and vtc2-4 mutants were observed. Unlike vtc2-1, the vtc2-4 mutants had wild-type zeaxanthin contents. While both low ascorbate mutants had lower levels of non-photochemical quenching of chlorophyll a fluorescence (NPQ) than the wild type under HL, qPd values were greater only in vtc2-1 leaves. Ascorbate is therefore essential for growth but not for photoprotection

(Homo)glutathione Deficiency Impairs Root-knot Nematode Development in Medicago truncatula

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes

Anthocyanin biosynthesis mystery in gerbera cultivars Estelle and Ivory

Flavonoids in our model ornamental plant Gerbera hybrida, consist of three subgroups, flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial surface of petals and give the different cultivars their different color. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. The acyanic cultivar Ivory is a sport of the pelargonidin containing pink cultivar Estelle, i.e., it originates from an acyanic branch of Estelle. Ivory is apparently a transposon mutant of Estelle, since revertant sectors are regularly observed (Figure 1). In spite of complete loss of anthocyanin pigmentation, all genes encoding enzymes necessary for pelargonidin biosynthesis (PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, DFR, and ANS) are expressed in Ivory at similar levels as in Estelle. We performed a comprehensive flavonoid analysis using UHPLC MS/MS for Estelle and Ivory, collecting samples from whole ray flower petals and from their isolated adaxial epidermi. Except for pelargonidin derivatives, which are present in Estelle but lack nearly completely from Ivory, we found that both cultivars have similar amounts of flavones and flavonoids (mainly apigenin and kaempherol glycosides). We further analyzed the cultivars using RNA sequencing and produced on average 10 million Illumina reads from two developmental stages of Estelle and Ivory petals. Mapping of the reads to an assembly of gerbera Sanger (1), 454 and Illumina reads confirms that all anthocyanidin biosynthesis genes are expressed similarly in the two samples. Surprisingly, none of the assembled contigs show differential expression between these two cultivars. Although dramatically different to the eye, the difference in Estelle and Ivory at transcript level eludes our attempts of analysis. Pelargonidin biosynthesis is intact at least up to the point of dihydrokaempherol synthesis. In reads mapping to transcripts for DFR and ANS we have not observed anomalies that would be indicative of a transposon insertion. None of the glucosyl transferase encoding transcripts are down regulated or anomalous either. However, we do not have biochemical evidence which one of the contigs would encode the gerbera anthocyanidin 3-O-glucosyltransferas

Structural analysis of two distinct dihydroflavonol 4-reductases in Gerbera Hybrids

Dihydroflavonol 4-reductase (DFR) is a key enzyme within anthocyanin biosynthesis known for its distinct substrate specificity found in various plant species (1,2). Based in the findings of enzymatic studies and in vivo inhibition experiments in cyanidin accumulating genotype “Clivia” two DFRs with different substrate preference were postulated. Cloning approaches with petals of “Clivia” led to the identification of a second DFR sequence sharing around 96% identity to the previously cloned DFR from pelargonidin accumulating variety “Regina” (1,3). The obtained recombinant protein shows a higher preference for dihydroquercetin (DHQ) while dihydrokaempferol (DHK) was still converted to leucopelargondin (LPg) to a certain extend. However, the overall ratio showed a clear preference for the cyaniding branch of the pathway. To shed light on this biochemical aspect of the anthocyanins biosynthetic pathway, we have attempted an in silico structure-based approach aiming to relate the specific amino acid differences in the DFRs of Gerbera “Regina” and “Clivia” varieties, with their molecular structures and enzyme selectivity data. The DFR models have been built using the crystal structure of the DFR from Vitis vinifera4 (PDB ID 2C29) as a template. We suggest that a residue belonging to the “specificity loop” located near the substrate “binding site” confers the observed substrate-binding specificity. Namely, Gly135 (Gerbera “Regina” DFR) or Val135 (Gerbera “Clivia” DFR) respectively, is likely to unlock or lock the orientation of the conserved residue Asn134 that in turn is engaged in hydrogen bonding interactions with 4’ OH’ of ring B of DHK or both 3’ OH’ and 4’ OH’ of ring B of DHQ. Interestingly the side chain orientation of Val135 (Gerbera “Clivia” DFR) is restricted and stabilized by hydrophobic interactions with the conserved residues Ile154 and Phe165