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See what Riley (MsWendy_Playz) has discovered on Pinterest, the world's biggest collection of ideas.uA 'aa tnd aints eqlo amcr no dJ, en-1 eptune ii ninea Consu.- MI lt III Nocllot mes PIL ,LI IUU U $25. o 3.qU R I-I 15 Sisuen ess I a m y le Sorehin rr i. ueusioll is "d"idl sass dol Is sasa eLaHbas Id ulado148 eba n dongea aitist o ll- stA5 ad Prd bt-127 lb,, Sai o r Ca.i IL ~ fl U V's'2 Uria h ec aled ur udr iy smdcPurification of glutathione-binding proteins from pigmented V. vinifera cell suspension cultures. (A) Vitis vinifera FU-01 cells were cultured in GC-2 medium (filled circles) for 4 d, then elicited with 10 μM jasmonic acid, 20 g l −1 sucrose (or an equal volume of vehicle control), and constant white light irradiation (open circles, 96.8±2.2 μmol s −1 m −2)..15rS9 "El porifolillsitl t1r. 121 aiios al servicing de Jos intepm en lo ex 4140 ling profe on, en lo inter"'o reses generals v pernianentes un sacerditicio".Breeding for strong red skin color is an important objective of the pear breeding program. There are few reports of proteome research in green skin pear and its red skin bud mutation. The manuscript at hand is one of the first studies dealing with 2D-PAGE-based analysis of pear fruits and leaves, establishing a suitable sample preparation and testing different 2D-PAGE protocols.

Diario de la marina - UFDC Home

Browse by Name. Browse for your friends alphabetically by name. Numbers 0 to 25 contain non-Latin character names. Note: This only includes people who have Public Search Listings available on Facebook.UNK the , . of and in " a to was is ) ( for as on by he with 's that at from his it an were are which this also be has or : had first one their its new after but who not they have - ; her she ' two been other when there all % during into school time may years more most only over city some world would where later up such used many can state about national out known university united then madeTotal protein was extracted from c. 50 g of day 2-3 B. calycina petals with a citrate phosphate extraction buffer (20 mM citrate buffer, pH 5, 10 mM CaCl 2, 5 mM DTT, 10 mM thiourea, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.025 g ml −1 polyvinylpolypyrrolidone (PVPP)).Pear is a widely grown and popular nutrient-rich fruit whose ripening mechanism was elucidated by 2-DE (Hu et al., 2012), 2D-DIGE (Gao et al., 2016), iTRAQ labeling and label-free (Reuscher et al

Diario de la marina - UFDC Home

Purification, molecular cloning, and characterization of

ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz~ € ‚ƒ„…†‡ˆ‰Š‹Œ Ž ''""•-—˜™š›œ žŸ ¡¢£¤¥¦§¨©ªApproximately 1.5-2 g of pear leaves or fruits mixed in PVPP, according to the proportion of 1/5, and 0.1 g DTT were ground to a fine powder in liquid nitrogen with mortar and pestle. Each microgram of protein powder precipitated with 15 μl of cold phenol extraction buffer [7 mol/l urea, 2 mol/l theorem, 4% (w/v) CHAPS, 65 mmol/l DTT, 0.2% (vSo again on the trail of past year's time on my blog and as a thank you for all my readers and followers I am doing this short post. 2019 was very special ;my first full year alone with my boys having lost my dear late wife Martine in 2018. Time they tell me soothes the pain, actually is the other way around but we go on with responsabilities and challenges into 2020 probably my last fullHello Switzerland is written by expats for expats living in Switzerland. Designed mainly for English speakers, the magazine contains features, articles and information to help expats feel at homeDJ Marketing Bud's Salads DoorDash, Inc. Dot-It Restaurant Fulfillment DR Delicacy Fresh Truffles & Mushrooms Dr Pepper Snapple Group Dr. Praeger's Purely Sensible Foods Dripping Springs Distilling and Bloody Revolution DTT Dump Dolly LLC SPOONY! Dunbar Armored Dynamic USA East Montgomery County Gamay Food Ingredients Garcia Foods, LLC

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In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers - Zipor - 2015 - New Phytologist

Introduction

Anthocyanins are the largest group of plant pigments responsible for the red, violet and blue colors of flowers, fruit, seeds and vegetative tissues. They are ubiquitous in plants and elucidation of their roles is revealing their vital importance for the survival of plants in their environments. Apart from their obvious attractant or repellent effects on pollinators/dispersers or herbivores, anthocyanins seem to act as phyto‐protectors for a multitude of stress effects, of which protection from excess UV light is the best characterized (Chalker‐Scott, 1999). Anthocyanins are also of great economic importance. They are widely used as natural food colorants, they are extremely important for floriculture and they are thought to have numerous health benefits (Butelli et al., 2008).

Anthocyanins are soluble phenolic compounds that accumulate in the cell vacuoles, mainly in the epidermal and subepidermal tissues. Anthocyanin aglycones are comprised of two aromatic rings and an oxygen‐containing heterocyclic ring. These aglycones form a small group that is modified by the addition of glycosyl and aromatic or aliphatic acyl moieties to yield hundreds of anthocyanins (Anderson & Jordheim, 2006). The biosynthetic pathway of anthocyanins and its regulation have been characterized in detail, and this knowledge has led to the production of transgenic plants with altered composition of anthocyanins (Quattrocchio et al., 1998; Bradley et al., 2000; Davies et al., 2003; Ben Zvi et al., 2008; Butelli et al., 2008; Tanaka & Ohmiya, 2008). However, in contrast to extensive knowledge regarding their biosynthesis, very little is known about the degradation process of anthocyanins in living plant tissues, a factor that should also be taken into account when seeking to produce plants with altered concentrations of anthocyanin.

Anthocyanins are degraded in plant tissues at specific developmental stages or as a consequence of changes in environmental conditions (Oren‐Shamir, 2009). This degradation can be explained as a consequence of their roles as signals for pollinators in flowers, for seed dispersers in fruits, and as protectors of photosynthetic tissues from damaging light (Steyn et al., 2002). For example, the anthocyanins that accumulate in the epidermal tissues of young developing foliage serve as ‘sunscreens’ that protect the leaves from damaging high light intensities. As the leaves mature and develop protective waxes, the anthocyanins are degraded, thus enabling more light penetration to the photosynthetic apparatus (Oren‐Shamir & Nissim‐Levi, 1999; Nissim‐Levi et al., 2003). Similarly, anthocyanins are degraded in fruit peels as a result of increased temperature or decreased light intensity, enabling more light to penetrate the developing tissues (Steyn et al., 2004). Anthocyanin degradation as a result of decreased light intensities and increased temperatures occurred even in red‐pigmented Arabidopsis thaliana plants overexpressing the gene encoding the Production of Anthocyanin Pigment 1 (PAP1) MYB transcription factor (Rowan et al., 2009). The loss of red pigmentation in A. thaliana plants overexpressing PAP1 was accompanied by reduced expression of several genes in the PAP1 complex and by increased expression of potential transcriptional repressors (Rowan et al., 2009). These examples suggest that anthocyanin degradation is controlled and induced when beneficial to the plant, implying involvement of enzymatic activity in the process. In addition to specific degradation, there may be turnover of the pigments in plant tissues, even when no visual change in color is observed. This may be a result of either chemical instability of the pigments or regulated enzymatic activity for precise spatial and temporal fine‐tuning of the concentrations of the pigments (Oren‐Shamir, 2009; Sinilal et al., 2011).

Proof of active in planta anthocyanin degradation in plants was obtained in flowers of Brunfelsia calicina (Solanaceae) (Vaknin et al., 2005). These flowers change color from dark purple to pure white within the first 2 d after opening and well before the onset of senescence. This process was shown to be dependent on anthocyanin degradation and de novo synthesis of mRNAs and proteins (Vaknin et al., 2005). Brunfelsia calicina is a unique system for studying the enzymatic process of anthocyanin degradation as this process results in a dramatic and rapid color change of the flowers, and occurs at a specific and well‐defined stage of flower development (Vaknin et al., 2005; Bar‐Akiva et al., 2010). Identification of the anthocyanin‐degrading enzymes in B. calicina may lead to a general understanding of how this process occurs in other plants and plant organs during the fine‐tuning and dynamic regulation of the concentrations of these compounds, which is of great importance for the interaction of plants with an ever‐changing environment. Moreover, this knowledge is extremely important for the manipulation of flower color, with great potential for economic applications.

The enzymatic process by which anthocyanins are degraded in planta is still not known. However, previous studies in fruit extracts and juices provide a basis for understanding in planta anthocyanin degradation. Three enzyme families were shown to be involved in this process in fruit juices: polyphenol oxidases (PPOs), class III peroxidases and β‐glucosidases (Oren‐Shamir, 2009). One possible pathway for enzymatic anthocyanin degradation in fruit extracts is the reduction of quinones parallel to oxidation of anthocyanins either by PPOs or peroxidases. Another possibility is a two‐step degradation, including deglycosylation of sugar moieties and then oxidation of the aglycone by either PPOs or peroxidases (Oren‐Shamir, 2009).

Of the three enzyme families, peroxidases and β‐glucosidases are more likely candidates for in planta anthocyanin degradation, as they are frequently found in the vacuoles. All known PPOs are located in the plastids, with one exception of an aurone biosynthetic enzyme suggested to be vacuolar (Ono et al., 2006).

Vacuolar peroxidases, belonging to the class III peroxidase family, are able to catalyze the reduction of toxic H2O2 that reaches the vacuoles by oxidizing a variety of secondary metabolites (Ferreres et al., 2011). Despite the importance of these enzymes, current understanding of their roles in the plant cell is very limited and based on incomplete and correlative data (Zipor & Oren‐Shamir, 2013). Vacuolar peroxidases seem to be involved in different aspects of plant adaptation to change, including scavenging of hydrogen peroxide, which accumulates under stressful conditions, and synthesis of phytoalexins, which accumulate following pathogen attack (Zipor & Oren‐Shamir, 2013). A vacuolar peroxidase has also been implicated in the biosynthesis of defense alkaloids in the medicinal plant Catharanthus roseus (Costa et al., 2008). The possible involvement of vacuolar peroxidases in anthocyanin degradation may be a component of the adaptation of plants to changing environmental conditions such as decreased light intensities, but may also be a component of the plant developmental program, as seems to happen in Brunfelsia flowers.

Previously, we demonstrated a dramatic increase in total peroxidase activity in Brunfelsia flower petals in correlation with the onset of anthocyanin degradation (Vaknin et al., 2005). Here, we show that a single peroxidase, BcPrx01, located in the vacuole and induced after flower opening, is responsible for the degradation of anthocyanins in the flowers. In vivo anthocyanin‐degrading enzymes have not been characterized in plants to date. Our findings suggest that vacuolar peroxidases play a key role in the regulation of in vivo anthocyanin concentration.

Materials and Methods

Plant material and growth conditions

Brunfelsia calycina (Hook.) Benth. seedlings were grown in 18‐cm pots in a glasshouse. For flower induction, 1–3‐yr‐old plants were transferred to a 17°C : 9°C regime (day : night, respectively) for a period of 9 wk in natural daylight conditions. After flower bud formation, plants were transferred to an air‐conditioned glasshouse with maximum temperatures not exceeding 20°C, with long day (LD) conditions (16 h light). Flowers were collected from the plants at the time of bud opening (day 0) and on specific days before and after opening (between days ‐1, 1, 2, 2‐3, 3 and 4). Petunia hybrida line V26 plants were grown in pots in an air‐conditioned glasshouse at 22°C with LD conditions (16 h light).

Peroxidase enrichment and activity

Total protein was extracted from c. 50 g of day 2–3 B. calycina petals with a citrate phosphate extraction buffer (20 mM citrate buffer, pH 5, 10 mM CaCl2, 5 mM DTT, 10 mM thiourea, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.025 g ml−1 polyvinylpolypyrrolidone (PVPP)). Soluble proteins were purified from crude extract and PVPP by precipitation at 13 000 g for 30 min and filtration. Proteins were precipitated using 30–100% ammonium sulfate. The pellet was resuspended and dialyzed against 20 mM citrate buffer, pH 5, and loaded on a cation exchange 1‐ml SP Sepharose HP column (GE Healthcare, Little Chalfont, UK), using a 30‐cv gradient (1 M NaCl). One‐milliliter fractions were collected and assayed for protein concentration and peroxidase activity. Protein concentration was quantified using a BCA protein assay kit (Pierce, Thermo Scientific, Waltham, MA, USA). Peroxidase activity was measured in a 10 mM sodium acetate butter at pH 4, containing 0.4 mM 3,3′,5,5′‐tetramethylbenzidine (TMB) and 3 mM H2O2 (Josephy et al., 1982). Activity was measured at 25°C in an enzyme‐linked immunosorbent assay (ELISA) plate scanner (Spectra MR; DYNEX Technologies, Denkendorf, Germany) at absorbance 450 nm.

Semi‐native PAGE, isoelectric focusing (IEF) and staining of peroxidase acivity

Separation of peroxidase isozymes was performed by mixing enzyme extracts with glycerol and bromophenol blue and loading on a 10% polyacryl amide gel with 0.1% SDS. Staining of the gels for peroxidase activity was performed according to Andrews et al. (2000). IEF and determination of IEF in‐gel peroxidase activity, using 4‐methoxy‐α‐naphthol (4‐MN), were performed according to Ferreres et al. (2011).

Glucosidase activity

β‐glucosidase activity of day 0 and day 2–3 total proteins of B. calycina was measured according to van Tilbeurgh et al. (1988) with the following modifications. Samples were incubated for 1 h in the dark at 37°C in a 10 mM sodium acetate butter, pH 5, containing 5 mM 4‐methylumbelliferyl β‐d‐glucopyranoside (MUGlc). Different amounts of d‐gluconic acid, a β‐glucosidase inhibitor, were added as described in the 3 section. The reaction was stopped using 0.2 M sodium carbonate. Fluorescence emission was measured at 460 nm after excitation at 360 nm.

Anthocyanin degradation assay

Anthocyanins from P. hybrida and B. calycina petals were extracted according to Markham & Ofman (1993). The extract was dried and resuspended in 10 mM sodium acetate buffer for pH values between 3 and 6, and Tris–HCl buffer for pH 8. Degradation assays were carried out at room temperature (RT) in 1‐ml cuvettes in a spectrophotometer (UV‐2401PC; Shimadzu, Kyoto, Japan) at 539 nm (maximum peak of P. hybrida anthocyanin extract). For LC‐MS analysis of the change in anthocyanin composition during degradation, anthocyanins were dried in a speedvac at RT (Savant SPD111V; Thermo Scientific) and resuspended in formic acid solution, pH 4. LC‐MS analysis of the pigments was carried out as described in Sinilal et al. (2011). Characterization of the anthocyanins was based on measurement of multiple accurate mass MS fragments (Ando et al., 1999).

Protoplast isolation

Cell wall digestion enzymes (1% cellulase ‘Onozuka’ R‐10, 0.2% macerozyme R‐10, and 0.4% driselase) were prepared in CPW 9 M salts buffer containing 11% mannitol. Petals from day 2–3 B. calycina were cut into 1‐mm strips inside the digestion buffer and incubated at 25°C in the dark for c. 4 h (until round cells were disengaged from the tissue). The liquid was filtered through nylon mesh (100 μm) followed by protoplast centrifugation at 70 g for 5 min at 25°C. Viability of protoplasts was tested with 2 μg ml−1 fluorescein diacetate (FDA) using a fluorescence microscope (green filter). Proteins were extracted by liquid nitrogen freezing followed by adding citrate phosphate buffer (20 mM citrate buffer, pH 5, 10 mM CaCl2, 5 mM DTT, 10 mM thiourea, and 1 mM PMSF), and analyzed for the peroxidase profile.

Cytochemical localization of peroxidase‐like activity

Day 2 petals of B. calycina were cut into 1‐mm strips. The strips were fixed and stained with 3,3′‐diaminobenzidine (DAB) as described by Ferreres et al. (2011). Controls were performed with the same reaction mixture without DAB. Samples were analyzed using standard transmission electron microscopy (TEM) procedures.

RNA extraction, sequencing and assembly

Total RNA of day 0 and day 2 B. calycina flowers was extracted using the ZR plant RNA miniprep (R2024; ZYMO Research, Irvine, CA, USA), and further treated with DNase I (#EN0525; Fermentas, Vilnius, Lithuania). Two microliters of day 0 and day 2 (two biological repeats) total RNA was prepared for sequencing using the Sample prep‐TruSeq RNA kit from Illumina (San Diego, CA, USA), according to the manufacturer's instructions. The sequencing was performed using Illumina HiSeq 2500 rapid mode 2 × 100 bp paired‐end sequencing including one barcode with onboard clustering. Transcriptome de novo assembly was performed using trinity software (version trinityrnaseq_r2013_08_14; Grabherr et al., 2011). The distinct transcripts were used for blast searches (Altschul et al., 1990) and annotation against four protein databases: the tomato (Lycopersicum esculentum) protein sequences database (http://solgenomics.net/ version ITAG2.3), the TAIR database (version TAIR10; http://www.arabidopsis.org), the Uniref90 sequence database (Suzek et al., 2007), and the PeroxiBase database (Fawal et al., 2013). Best hits with an E‐value of e−5 were considered significant.

Differential expression analysis

The TopHat (version v2.0.6) and Cufflinks (v2.1.1) software tools (Trapnell et al., 2012) were used to map the reads to the reconstructed transcriptome. Then, Cuffdiff software (version v2.1.1) was used to test for significant differential expression between day 0 and day 2 with a false discovery rate (FDR) threshold of 0.05 (Li et al., 2012).

Shotgun proteomic analysis

Total protein was extracted from day 0 and day 2 B. calycina petals with citrate buffer as described above. The proteins were dialyzed against H2O. 7.5 μg protein samples were then subjected to in‐solution digestion (four biological repeats per group). Proteins were first reduced using 5 mM dithiolthreitol (Sigma) for 30 min at 60°C, followed by alkylation with 10 mM iodoacetemide (Sigma) for 30 min in the dark. Sequencing grade trypsin (Promega, Madison, WI, USA) was added at a ratio of 50 : 1 (protein : trypsin) and incubated at 37°C overnight. Digestion was stopped by the addition of 1% trifluroacetic acid. Peptide mixtures from each sample were analyzed by label‐free shotgun proteomics using two‐dimensional liquid chromatography coupled with tandem high‐resolution mass spectrometry. Samples were run in a random order. Raw data were aligned and quantitative information extracted using the Rossetta Biosoftware Elucidator system (Seattle, WA, USA) as described by Levin et al. (2011). MS/MS spectra were searched using Proteinlynx Global Server (plgs) version 2.5.2 (Waters, Milford, MA, USA) against a transcriptome assembly obtained using GeneMark™ software (Besemer & Borodovsky, 1999). Protein quantification was obtained based on the three most abundant peptides per protein (Silva et al., 2005).

Phylogenetic analysis of peroxidase homologs

Alignment with 12 peroxidase sequences was performed using the muscle program using default parameters (http://www.ebi.ac.uk/Tools/msa/muscle/; Edgar, 2004).

A phylogenetic tree was reconstructed based on a maximum likelihood (ML) framework using PhyML software (Guindon & Gascuel, 2003) based on the LG matrix‐based model (Le & Gascuel, 2008). The tree was graphically designed using FigTree version 1.4 (http://tree.bio.ed.ac.uk/software/figtree/).

In gel proteolysis and mass spectrometry analysis

The proteins in the gel were reduced in 5 mM DTT solution at 60°C for 30 min, modified in 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate at RT in the dark for 30 min, and digested in 10 mM ammonium bicarbonate, 10% acetonitrile and modified trypsin (Promega) overnight at 37°C. The resulting tryptic peptides were resolved by reverse‐phase chromatography on 0.075 × 200‐mm fused silica capillaries (J&W, Folsom, CA, USA) packed with Reprosil reversed‐phase material (Dr Maisch GmbH, Germany). The peptides were eluted for 105 min in a linear gradient of 5–28% acetonitrile with 0.1% formic acid in water, 15 min in a gradient of 28–90% acetonitrile with 0.1% formic acid in water, and 15 min in 90% acetonitrile with 0.1% formic acid in water at a flow rate of 0.15 μl min−1. Mass spectrometry was performed using an Q‐Exactive mass spectrometer (QE, Thermo Fisher) in a positive mode using a repetitive full MS scan followed by high‐energy collision dissociation (HCD) of the 10 most dominant ions selected from the first MS scan. The mass spectrometry data were analyzed using Protein Discoverer 1.4 software with Sequest (Thermo) algorithms.

Accession codes

Transcriptomic data for day 0 and 2 B. calycina flowers were deposited in GenBank with BioProject ID PRJNA229456, and the raw reads were submitted to the Sequence Read Archive (SRR: day0 SRR1037136; day2a SRR1037137; day2b SRR1037138). The accession number for BcPrx01 is KF798194.

Results

Peroxidase activity in B. calycina flowers mediates in vitro anthocyanin degradation

Anthocyanin degradation in B. calycina flowers is an active enzymatic process, dependent on the induction of genes and on synthesis of a novel protein(s) after flower opening (Vaknin et al., 2005). To test the ability of B. calycina enzymes to degrade anthocyanins, an assay was developed in which a total protein extract from B. calycina was applied to P. hybrida anthocyanins, and the ability of the extract to degrade anthocyanins was followed over time. The proteins were extracted from B. calycina flowers at days 2–3 after flower opening, when the rate of depigmentation of the flowers is maximal (Vaknin et al., 2005). The anthocyanins were extracted from P. hybrida line V26 flowers; flowers from this line were selected as they have a much higher pigment concentration than those of B. calycina, and a very similar pigment composition (Bar‐Akiva et al., 2010).

Peroxidase activity was found to be essential for the degradation of anthocyanins. When the total protein extract from flowers at day 2–3 was added to the pigment mixture, no significant degradation occurred. Only after addition of H2O2, which is required for peroxidase activity, were anthocyanins degraded (Fig. 1a). This degradation process occurred at a wide range of pH values, with the highest activity rate at pH 4 and only 50% of that activity at pH 8 (Fig. 1b). The maximum activity rate at pH 4 suggests that the anthocyanin‐degrading peroxidases may be active in the acidic cell vacuoles.

Degradation of anthocyanins mediated by peroxidase activity in Brunfelsia calycina day 2–3 flowers. (a) Degradation rate of anthocyanins measured upon addition of (1) 5 mM H2O2, (2) 5 μg ml−1 denatured day 2–3 B. calycina protein extract, (3) 5 μg ml−1 protein extract, (4) 5 μg ml−1 protein extract + 5 mM H2O2, and (5) 10 μg ml−1 protein extract + 5 mM H2O2. The degradation rate was determined by following the change in the maximal absorbance (A) wavelength of the extract (539 nm). (b) Degradation rate of anthocyanins in the presence of 10 μg protein extract + 5 mM H2O2 at different pH values. The values are means of three replicates ± SE.

Peroxidase degradation of anthocyanins was not specific to any of the anthocyanins in the mixture, as all components decreased in concentration during the degradation process (Fig. 2). This was true even though most of the anthocyanins characterized were complex, with several sugar and phenolic acid moieties attached to the aglycones (Fig. 2). Furthermore, no novel pigments were detected during the catabolic process, suggesting that the degradation is a one‐step process. Deglycosylation of the sugar moieties before the peroxidase activity would have resulted in formation of new anthocyanin components, before their oxidation and degradation. In addition, complete inhibition of glucosidase activity in the protein extract from day 2–3 flowers had no significant effect on the H2O2‐dependent ability of this extract to degrade anthocyanins (Fig. 3). Finally, unlike peroxidase activity, which increases in parallel to the anthocyanin degradation process in the flowers (Vaknin et al., 2005), the total glucosidase activity decreased in the flowers between day 0 and days 2–3 (Fig. 3a).

Change in the composition of anthocyanins during their degradation mediated by peroxidase activity from Brunfelsia calycina day 2–3 flowers. The different colored bands indicate the anthocyanins identified in the Petunia hybrida extract by LC‐MS analysis (partial identification). The ratio between the different anthocyanins was determined 100 and 300 s after addition of the B. calycina 10 μg ml−1 total protein extract from day 2–3 flowers and 5 mM H2O2. The identified anthocyanins were: petunidin 3‐caffeoylglucosylcaffeoylrutinoside‐5‐glucoside; malvidin 3‐caffeoylglucosyl p‐coumaroylrutinoside‐5‐glucoside; petunidin 3‐caffeoylglucosyl p‐coumaroylrutinoside‐5‐glucoside; petunidin 3‐glucosyl p‐coumaroylrutinoside‐5‐glucoside; petunidin 3‐cis‐p‐coumaroylrutinoside‐5‐glucoside; delphinidin 3‐rutinoside‐5‐glucoside; delphinidin 3‐rutinoside; delphinidin 3‐glucoside. Glucosidase activity in Brunfelsia calycina flowers and its effect on peroxidase‐mediated anthocyanin degradation. (a) Glucosidase activity was measured in total protein extracts (6.5 μg ml−1) from B. calycina flowers at day 0 and days 2–3, and in day 2–3 extracts in the presence of different concentrations of d‐gluconic acid, a glucosidase inhibitor. (b) The effect of total inhibition of glucosidase activity on the ability to degrade anthocyanins from a day 2–3 flower extract. The anthocyanin degradation rate was determined as ∆A539 μg−1 protein s−1. The values in (b) are means of three replicates ± SE.

The H2O2‐dependent ability of B. calycina flower proteins to degrade anthocyanins increased during flower development in parallel to the loss of color (Fig. 4). When total soluble proteins were extracted at different stages of flower development, and their ability to degrade anthocyanins in the presence of H2O2 was tested, a dramatic increase was observed, corresponding to the loss of flower color. The increase in the ability of the extract to perform peroxidase‐mediated degradation of anthocyanins began between days 1 and 2, similar to the in vivo degradation, and increased until day 4, when flowers became pure white (Fig. 4).

Increase in the ability of Brunfelsia calycina flower protein extracts to perform peroxidase‐mediated degradation of anthocyanins as the flowers develop and lose their pigmentation. The rate of anthocyanin degradation was determined at pH 4, in the presence of 5 mM H2O2, using 20 μg ml−1 total soluble protein extracts from days −1 to 4 of flower development. The anthocyanin degradation rate was determined by following the change in the maximal absorbance (A) wavelength of the extract (539 nm). The values are means of three replicates ± SE. Partial purification of BcPrx01, the peroxidase isoenzme that increases in activity parallel to in planta anthocyanin degradation

The peroxidase activity profile was compared between different B. calycina organs and flower developmental stages in peroxidase activity gels. The number of active peroxidase isoenzymes was smaller in the flowers in comparison to roots, stems and mature leaves (Fig. 5). The most conspicuous peroxidase isoenzyme observed was the main peroxidase present in day 3 flowers, which were undergoing color change. This isoenzyme also showed remarkable induction from day 0 to day 3 in flowers and was therefore clearly the most likely candidate for the peroxidase responsible for the observed H2O2‐dependent anthocyanin degradation (Fig. 5). Following the nomenclature used by PeroxiBase (www.peroxibase.toulouse.inra.fr) this peroxidase isoenzyme was named BcPrx01.

Profile of peroxidase isoenzymes from different Brunfelsia calycina organs obtained by semi‐native PAGE of nondenatured proteins. Three micrograms of total protein extract of different B. calycina organs and developmental stages was separated by semi‐native PAGE followed by 4‐chloro‐1‐naphthol staining. The arrow marks the molecular weight (MW) in kDa of the candidate anthocyanin‐degrading isoenzyme.

BcPrx01 was then partially purified from day 2–3 flowers by ammonium sulfate fractionation followed by cation exchange chromatography, to produce a fraction in which BcPrx01 was the single active peroxidase isoenzyme present (Fig. 6). The partially purified BcPrx01 fraction showed a 300‐fold increase in peroxidase specific activity, indicating a high degree of purification (Fig. 6c,d). Remarkably, the ability to degrade anthocyanins in the presence of H2O2 showed a commensurate or even higher increase in the enriched fraction of 400‐fold (Fig. 6d). Hence, BcPrx01 was the peroxidase isoenzyme responsible for the anthocyanin‐degrading activity detected in the day 2–3 flower extract.

The partially purified Brunfelsia calycina peroxidase 01 (BcPrx01) retains total anthocyanin‐degrading ability. (a) Total protein (280 nm) and peroxidase activity (450 nm) determined in cation exchange chromatography (CEC) fractions from a day 2–3 extract after ammonium sulfate fractionation. (b) In‐gel peroxidase activity from semi‐native PAGE of nondenatured proteins comparing equal protein amounts of the CEC fractions. (c) In‐gel peroxidase activity from semi‐native PAGE of nondenatured proteins from a total protein extract and fraction no. 1 from CEC. (d) Rate of anthocyanin degradation and peroxidase activity of the total protein extract and CEC fraction 1 containing only the BcPrx1 activity band. The values are means of three replicates ± SE. (e) An isoelectric focusing (IEF) gel of 1 μg of the purified fraction, stained with 4‐methoxy‐α‐naphthol. Molecular weight in kDa.

Further analyses of the peroxidase isoenzyme profile by IEF revealed that the main peroxidase isoenzyme induced at day 2–3 had a pI near 10, indicating that BcPrx01 is a basic peroxidase, similar to previously characterized vacuolar peroxidases (Fig. 6e) (Costa et al., 2008).

BcPrx01 co‐localizes with anthocyanins in planta

Assignment of a function to an enzyme is dependent on the confirmation of in vivo co‐localization with its putative substrate. Therefore, the subcellular localization of BcPrx01 was investigated using different methodologies. As class III peroxidases are secreted either to the cell wall or to the vacuoles (Cosio & Dunand, 2009), protoplast purification is sufficient to attribute the candidate peroxidase to one of the two organelles, and hence to determine its intracellular localization. The peroxidase activity profile was investigated in protein extracts from protoplasts isolated from day 2–3 B. calycina flower petals and from total proteins by semi‐native PAGE and IEF (Fig. 7). The peroxidase band previously identified as BcPrx01 was clearly the main if not the single peroxidase isoenzyme present in isolated protoplasts, strongly suggesting that this is the only active vacuolar peroxidase in the flowers during their change in color. The acidic isoenzymes induced between days 0 and 2–3 (Fig. 7c) were not present in the protoplast preparation, suggesting that these enzymes are in the cell wall.

Peroxidase activity in purified protoplasts. (a) Light microscopy photograph of protoplasts from day 2–3 Brunfelsia calycina flowers. (b) Fluorescein diacetate (FDA) staining of viable protoplasts. (c) Peroxidase activity gel of a protoplast preparation and total protein extract (10 μg) from day 2–3 flowers. (d) An isoelectric focusing (IEF) gel stained for peroxidase activity, comparing the protoplast preparation and total protein extract from day 0 and days 2–3. Arrows mark the two peroxidases with increased activity between day 0 and day 2–3. Molecular weight in kDa.

A direct indication of the presence of peroxidase activity in the vacuoles of B. calycina flowers was obtained by cytochemical localization of peroxidase using DAB and H2O2. H2O2‐dependent DAB oxidation formed black sediments in the inner part of the tonoplast, indicating the presence of peroxidase activity associated with the inner face of the vacuolar membrane (Fig. 8). Moreover, this activity was detectable in cells from the petals of flowers at days 2–3, when the BcPrx01 isoenzyme was highly induced, but not in petals at day 0 (not shown).

Peroxidase activity in day 2–3 Brunfelsia calycina petals. Transmission electron microscopy (TEM) analysis of day 2–3 petals was performed with (a) H2O2 and (b) 3,3′‐diaminobenzidine (DAB) + H2O2. Peroxidase activity is indicated in the black pigmentation in the inner part of the tonoplast, marked with arrows. The vacuolar region is marked ‘V’. Bar, 1 μm. Identification of the full‐length transcript sequence of the anthocyanin‐degrading BcPrx01

Attempts to identify the anthocyanin‐degrading transcript sequence by comparing LC‐MS/MS peptide sequencing of enriched protein extracts to known protein databases (such as NCBI) were unsuccessful. This was a consequence of the lack of a B. calycina database and the similarity between plant peroxidases. In order to identify the specific peroxidase induced after flower opening and responsible for the color change from purple to white, we performed an RNA‐Seq analysis (Supporting Information Table S1) comparing transcripts from day 0 and day 2. The transcriptomic data were used to produce a specific and comprehensive protein sequence database. From a total of c. 280 000 transcripts, four transcripts identified as class III peroxidases showed an increase in transcription level between day 0 and day 2 (Table 1; Fig. S1).

Table 1. Brunfelsia calycina flower peroxidase genes with increased expression between days 0 and 2 Peroxidase genea No. of identified amino acidsb mRNA (fold change, day 2 to day 0) Peptides in total extracts (fold change, day 2 to day 0) Peptides in peroxidase‐enriched fraction (fold change, day 2 to day 0) 56321 (BcPrx01) 359 20.00 Detected only on day 2 (one identified peptide)c 12.00 (11 identified peptides) 70671 324 1.42 2.42 (two identified peptides) ND 53337 266 3.90 ND ND 39892 231 2.94 ND ND 58911 320 ND ND Detected only on day 2 (one identified peptide) Coding sequences were deduced from the transcripts assembly using the GeneMarker software. Peroxidase transcripts induced on day 2 (q value < 0.05) were calculated using the differentially expressed gene (DEG) method (see the2 section). Peroxidase peptide amounts in the total protein extracts from day 0 and day 2 were compared using label‐free quantitative LC‐MS/MS proteomics, and are an average of four biological repeats. The numbers of peroxidase peptides in the day 0 and day 2 enriched fractions were determined by LC‐MS/MS (see text). ND, not detected. a Peroxidases have arbitrary names according to trinity software component numbering. b CDS were identified using the GeneMarker software. c Day 0 was below the detection threshold. The software estimated the increase as 647‐fold (P = 0.061).

Comparison of peroxidase peptide sequences obtained from LC‐MS/MS analysis of BcPrx01 activity bands enabled clear identification of transcript 56321 as the BcPrx01 gene: BcPrx01 was the only peroxidase identified in the activity gel band of the protoplast sample (Fig. 7c) and the IEF cationic band (Fig. 7c, arrow 1). In addition, BcPrx01 was the main peroxidase in the activity band of the peroxidase‐enriched fraction with the anthocyanin‐degrading capability (Fig. 6c, Table 1).

Analysis of the BcPrx01 deduced amino acid sequence and predicted three‐dimensional structure

The deep sequencing data enabled the identification of the full transcript sequence of BcPrx01 (Fig. 9a), which was later verified by standard sequencing of the RT‐PCR amplified product using specific primers designed to amplify the whole cDNA. The open reading frame (ORF) of the full‐length cDNA corresponds to a deduced polypeptide of 359 amino acids. The polypeptide includes a putative signal peptide (N‐terminal pro‐peptide) of 20 amino acids (MACGATHVSLALSLLALTLA), as identified using the SignalP software (Petersen et al., 2011). The signal peptide, common to all class III peroxidases, directs the polypeptide to the endoplasmic reticulum (ER) membrane. The polypeptide also includes the conserved peroxidase active site signature (93‐GASLIRLFFHDC) and a peroxidase proximal heme‐ligand signature (223‐EMVALAGAHTI). The predicted MW of the full‐length polypeptide is 38.6 kDa. The theoretical pI of the mature protein is 6; however, the IEF gels clearly demonstrated its basic nature, having a pI of c. 10 (Figs 6e, 7d). The presence of BcPrx01 in the basic band in the IEF gel was verified by direct LC‐MS/MS sequencing. To characterize BcPrx01 further, a hypothetical 3D structure was constructed using swiss‐model (Guex & Peitsch, 1997). The predicted spatial structure of BcPrx01 indicates a substantial presence of positive electrostatic potentials in the surface area, illustrating its basic nature (Fig. 9b).

Identification of the Brunfelsia calycina anthocyanin‐degrading peroxidase (BcPrx01). (a) Nucleotide and deduced amino acid sequence. Putative domains include: a signal peptide (purple), a peroxidase active site signature (green), a peroxidase proximal heme‐ligand signature (yellow), and a C‐terminal extension (broken underline in blue). Peptides that were identified in the peroxidase‐enriched fraction by LC‐MS/MS are underlined in red. (b) Hypothetical 3D structural model of BcPrx01 predicted by swiss‐model and viewed using Swiss‐PdbViewer 4.1.0 (Swiss Institute of Bioinformatics, Lausanne, Switzerland). Colors represent surface charge: white, neutral; blue, positive; and red, negative. The protein was taken to be at pH 7.0 for the calculation of the electrostatic potential. (c) Unrooted neighbor‐joining phylogenetic tree relating BcPrx01 to other class III peroxidases. The following sequences retrieved from PeroxiBase were used: (1) highly identical peroxidases; (2) extensively studied basic peroxidases (barley BP1, horseradish PrxC1 and C. roseus CrPrx1); (3) seven further Arabidopsis thaliana peroxidases, representing the seven branches revealed in the phylogenetic tree of A. thaliana peroxidases constructed by Tognolli et al. (2002). The nomenclature is according to class III PeroxiBase (http://peroxidase.isb-sib.ch). Phylogenetic analysis

BLASTp search analysis of the amino acid sequence deduced for BcPrx01 using PeroxiBase (Swiss Institute of Bioinformatics Blast Network Service; http://peroxidase.isb-sib.ch) revealed the highest homology with NtPrx32 from tobacco (Nicotiana tabacum) and LePrx21 from tomato (Lycopersicum esculentum). The A. thaliana peroxidase protein with the highest identity to BcPrx01 is peroxidase 52 (NP_196153). To elucidate the phylogenetic relationships of BcPrx01, an unrooted neighbor‐joining phylogenetic tree was constructed including: (1) highly identical peroxidases from tobacco, tomato and A. thaliana; (2) extensively studied vacuole‐localized peroxidases from C. roseus, barley (Hordeum vulgare) and horseradish (Armoracia rusticana); and (3) seven more A. thaliana peroxidases, representing all seven branches revealed in the phylogenetic tree of A. thaliana peroxidases constructed by Tognolli et al. (2002). The constructed tree (Fig. 9b) shows that BcPrx01 is phylogenetically related to the highly identical peroxidases and to A. thaliana peroxidases AtPrx52 and AtPrx40, and shares a common ancestor with PrxC1a (a horseradish peroxidase), a vacuolar peroxidase. Together, these peroxidases are on a separate phylogenetic branch from other A. thaliana peroxidase groups, as well as from two other vacuolar peroxidases, Barley Peroxidase 1 (BP1) and Catharanthus roseus Peroxidase 1 (CrPrx1).

Expression profile of BcPrx01

The transcriptomic data revealed four peroxidases with increased expression between days 0 and 2 of flower development (Table 1). Of the four transcripts, BcPrx01 expression increased 20‐fold, while the fold increase of the other three genes was between 1.4 and 3.9.

Peroxidase peptides that increased in their expression levels between days 0 and 2 were identified by comparing the peptide sequences obtained from LC‐MS/MS analysis to the RNA‐Seq data. Comparison of peroxidase levels in total protein extracts from days 0 and 2 was performed using shotgun proteomic analysis (see the 2 section). Two peroxidases with increased expression levels were identified. One of these peroxidases was BcPrx01, with one peptide detected only on day 2. The second peroxidase corresponds to transcript 70671, with two identified peptides (Table 1). Comparison of peroxidase levels from the peroxidase‐enriched fractions from days 0 and 2 (Fig. 6) was performed by comparing the proteins from in‐gel digestions of specific peroxidase activity bands by LC‐MS/MS analysis. Two peroxidases were identified, one of them being BcPrx01, with a 12‐fold increase and 11 identified peptides, and the second being 58911, with only one identified peptide.

The 70671 peroxidase was identified as the anionic enzyme that increased between days 0 and 2 (Fig. 7d, arrow 2), but was not detected in the protoplast fraction. The 53337 and 39892 proteins were not identified in any of the peroxidase activity bands correlating to anthocyanin degradation (Tables 1, 2). Peroxidase 58911 was only detected as a single peptide in the enriched fraction.

Table 2. Identification of peroxidases induced between days 1 and 2–3 in Brunfelsia calycina flowers in the isoelectric focusing (IEF) and protoplast activity gels Gene name Cationic band in IEF gel Anionic band in IEF gel Protoplast‐enriched gel band 56321 + − + 70671 − + − 58911 − − − 53337 − − − 39892 − − − The peroxidases induced on the second day in the IEF (marked in Fig. 7 as cationic (1) and anionic (2)) and in the protoplast‐enriched sample gel bands (Fig. 8) were subjected to LC‐MS/MS sequencing. Identified peroxidases are indicated as +.

In summary, all transcriptomic and proteomic data indicated a dramatic induction of the transcript and protein levels of BcPrx01 in flowers of B. calycina from day 0 to day 2 after opening, coincident with the occurrence of intense degradation of anthocyanins.

Discussion

Anthocyanins play a vital role in plant survival as attractants of pollinators/dispersers, repellents of herbivores, and phyto‐protectors in a variety of stress situations. To fulfill their role, they are often produced transiently, meaning that they undergo regulated degradation. However, although much is known about the biosynthesis of anthocyanins, very little is known about their degradation mechanisms in planta, in spite of their importance for the regulation of the final concentrations of these pigments.

Brunfelsia calycina flowers are a unique system for studying active in planta anthocyanin degradation, due to the specific and well‐defined developmental stages at which the catabolism is induced and anthocyanin degradation commences. Monitoring of degradation of the anthocyanins by the B. calycina protein extract enabled the in planta process to be mimicked, and revealed the fact that this process is dependent on a single vacuolar peroxidase. The detailed characterization of this system performed here clearly indicates that a novel vacuolar class III peroxidase isoenzyme, BcPrx01, is responsible for the in planta degradation of vacuolar anthocyanins during flower maturation in B. calycina.

The data show that BcPrx01 has the ability to degrade the complex anthocyanins in flowers, that it co‐localizes with these pigments in the vacuoles of petals, and that both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the massive degradation of anthocyanins occurring in B. calycina flowers. These results clearly confirm those of our early study describing the color change in B. calycina flowers (Vaknin et al., 2005), suggesting a strong control system.

Total inhibition of glucosidases in the B. calycina protein extract did not affect its ability to degrade the complex anthocyanins with several sugar moieties (Fig. 3), and this ability was retained in an extract in which the only active peroxidase was BcPrx01 (Fig. 6). Peroxidases have been shown to degrade anthocyanins in fruit extracts, provoked by exogenous application of H2O2. For example, a vacuolar peroxidase isolated from a Vitis vinifera cv. Gamay grapevine cell culture was shown to degrade grape anthocyanin aglycones such as peonidin, delphinidin and cyanidin, but not their glycosides, suggesting that this peroxidase may play a role in in vivo anthocyanin degradation (Calderon et al., 1992). Another example is increased anthocyanin degradation in aging strawberry (Fragaria ananassa) slices caused by the addition of exogenous H2O2, once again suggesting that peroxidase can be involved in the degradation of anthocyanins (Lopez Serrano & Ros Barcelo, 1999). Both cases suggest involvement of peroxidases in anthocyanin degradation, but do not imply that these enzymes can degrade anthocyanins on their own. The Gamay peroxidase is dependent on de‐glucosidation of the anthocyanins in order to oxidize the aglycones, and the strawberry peroxidase was only shown to influence the process that may involve glucosidases in the fruit slices.

Like other well‐studied vacuolar peroxidases, such as BP1 (Johansson et al., 1992) and CrPrx01 (Costa et al., 2008), BcPrx01 has a strongly cationic nature, as revealed by the IEF gels, with a pI of c. 10. Interestingly, the pI estimated for BcPrx01 based only on its sequence was 6. This difference was probably the result of the post‐translation modifications of peroxidases, including the binding of two calcium ions of the heme group, and the addition of N‐linked glycosydic chains at the ER (Welinder et al., 2002). Moreover, the BcPrx01 3D structure prediction (Fig. 9b) reveals the extensive presence of positive charges in the surface area of the protein, indicating that many of the acidic groups probably stay buried inside the protein, not contributing to its final pI.

The transcript sequence encoding the anthocyanin‐degrading enzyme, BcPrx01, was fully identified, and showed the highest identity with NtPrx32 (75%), LePrx21 (66%), and LePrx76 (60%). Curiously, LePrx76 has been suggested to be involved in cell wall suberization in tomato (Roberts & Kolattukudy, 1989). Costa et al. (2008) also observed that CrPrx01 did not seem to share similar functions with the peroxidases with higher identity, and suggested that multiplication of peroxidase genes preceded their recruitment for their present‐day functions. This is possible as a result of the overlapping reactivity properties of peroxidases that make them replaceable during evolution. Importantly, the phylogentic tree presented (Fig. 9c) also shows that BcPrx01 is related to the vacuolar horseradish peroxidase PrxC1a (Matsui et al., 2003), and to AtPrx40, which belong to the branch of A. thaliana peroxidases that includes all but one of the A. thaliana peroxidases predicted to be vacuolar (Tognolli et al., 2002).

Information on vacuolar peroxidases and their functions is still very limited (Zipor & Oren‐Shamir, 2013). Modification of a single peroxidase gene often results in no phenotypic effect, because of compensation by other redundant gene products. In addition, as most peroxidases can react with several substrates, in vitro experiments with a single enzyme rarely reveal the in planta substrate. For cell wall peroxidases, for example, there are only a few studies in which overexpression of the enzyme showed clear phenotypic effects of increased lignin content, demonstrating their in planta function (Lagrimini, 1991; Quiroga et al., 2000). However, there are no such studies of vacuolar peroxidases, and most of the knowledge about their function is from in vitro enzymatic activity studies, mainly at relatively low pH values, typical of vacuoles. To date, the functions of only two vacuolar peroxidases have been identified and characterized via in vitro experiments. One is the synthesis of the anticancer alkaloid anhydrovinblastine, by the vacuolar CrPrx1 in the leaves of the medicinal plant C. roseus (Sottomayor et al., 1998; Costa et al., 2008). The second is the oxidation of 3,4‐dihydroxyphenylalanine (DOPA) to the red‐brown DOPAchrome by a vacuolar peroxidase in broad bean (Vicia faba) (Takahama & Egashira, 1991). The anthocyanin‐degrading peroxidase in B. calycina flowers, BcPrx01, can now be added to the short list of characterized vacuolar peroxidases.

Anthocyanins are degraded in different plant organs under a variety of developmental and environmental conditions. In the vacuoles, degradation may be attributable to changes that decrease the stability of the pigments and cause either chemical degradation or increased vulnerability to degrading enzymes (e.g. β‐glucosidases and peroxidases) present in the vacuoles. However, there are examples, such as change in flower color as a signal for pollinators or loss of red pigmentation in photosynthetic tissues as a result of a change in environmental conditions, where it is clear that the degradation of anthocyanins is advantageous for the plants, and is probably controlled by specific genes and proteins (Oren‐Shamir, 2009).

In B. calycina, the dramatic change in color is accompanied by synthesis of fragrant phenolic volatiles. Both processes, anthocyanin degradation and synthesis of fragrant benzenoids, are probably signals for pollinators (Vaknin et al., 2005; Bar‐Akiva et al., 2010). The robust proteomic and transcriptomic data (Tables S1, S2) showed that many genes were differentially expressed between days 0 and 2, illustrating a tight regulation of the different processes occurring at this specific flower developmental stage. Further studies based on the B. calycina databases produced in this report may reveal the network of volatile and pigment phenolic metabolism in B. calycina and related species.

In conclusion, this work has shown that anthocyanin degradation in B. calycina flowers is probably mediated by a novel basic, vacuolar class III peroxidase, BcPrx01, whose gene was fully identified. This is a significant breakthrough in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been obtained, clearly indicating what may be a general role of vacuolar peroxidases. Moreover, this work unravels a clue regarding anthocyanin catabolism in plants. This is extremely relevant in many physiological states, particularly during the response to many types of stress, but is also very important for strategies aiming to increase the concentrations of economically desirable anthocyanins.

Acknowledgements

Many of the results presented in this paper were the fruits of a short‐term scientific mission (STSM), within COST Action FA1006. We would like to thank the Smoler Proteomics Centre in the Technion, Haifa, Israel for peptide sequencing and analysis.

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