Recombinant Plant Research Articles

Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy

The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive.In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in Pichia pastoris. We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80U/mg to more than 1000U/mg.

Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)n (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)6. The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)6 hydrolytic reaction was 3.9 at 37°C. The optimal temperature for activity was 65°C in 50mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid.

The influence of identity elements on the aminoacylation of tRNAArg by plant and Escherichia coli arginyl‐tRNA synthetases

Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyl-tRNA synthetases have been largely ignored in this respect. We have designed in vitro tRNA transcripts, based on the soybean tRNA(Arg) primary structure, aiming to investigate its specific aminoacylation by two recombinant plant arginyl-tRNA synthetases and to compare this with the enzyme from E. coli. Identity elements at positions 20 and 35 in plants parallel those previously established for bacteria. Cryptic identity elements in the plant system that are not revealed within a tRNA(Arg) consensus sequence compiled from isodecoders corresponding to nine distinct cytoplasmic, mitochondrial and plastid isoaccepting sequences are located in the acceptor stem. Additionally, it has been shown that U20a and A38 are essential for a fully efficient cognate E. coli arginylation, whereas, for the plant arginyl-tRNA synthetases, these bases can be replaced by G20a and C38 with full retention of activity. G10, a constituent of the 10:25:45 tertiary interaction, is essential for both plant and E. coli activity. Amino acid recognition in terms of discriminating between arginine and canavanine by the arginyl-tRNA synthetase from both kingdoms may be manipulated by changes at different sites within the tRNA structure.

Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism

Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

Mitochondrial Cysteine Synthase Complex Regulates O-Acetylserine Biosynthesis in Plants

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [(3)H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.

Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae)

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.

Assembly of Synthetic Locked Phycocyanobilin Derivatives with Phytochrome in Vitro and in Vivo in Ceratodon purpureus and Arabidopsis

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.

Structures of Hydrocarbon Hydrates during Formation with and without Inhibitors

The formation of hydrates from a methane-ethane-propane mixture is more complex than with single gases. Using nuclear magnetic resonance (NMR) and high-pressure powder X-ray diffraction (PXRD), we have investigated the structural properties of natural gas hydrates crystallized in the presence of kinetic hydrate inhibitors (KHIs), two commercial inhibitors and two biological ice inhibitors, or antifreeze proteins (AFPs). NMR analyses indicated that hydrate cage occupancy was at near saturation for controls and most inhibitor types. Some exceptions were found in systems containing a new commercial KHI (HIW85281) and a recombinant plant AFP, suggesting that these two inhibitors could impact the kinetics of cavity formation. NMR analysis confirmed that the hydrate composition varies during crystal growth by kinetic effects. Strikingly, the coexistence of both structures I (sI) and II (sII) were observed in NMR spectra and PXRD profiles. It is suggested that sI phases may form more readily from liquid water. Real time PXRD monitoring showed that sI hydrates were less stable than sII crystals, and there was a conversion to the stable phase over time. Both commercial KHIs and AFPs had an impact on hydrate metastability, but transient sI PXRD intensity profiles indicated significantly different modes of interaction with the various inhibitors and the natural gas hydrate system.

Recombinant plant made antibodies against fimbriae of enterotoxigenic Escherichia coli inhibit in vitro binding to villous enterocytes.

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Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

Environmental impact assessment and monitoring of genetically modified trees

Transgenic biotechnology can assist forest tree improvement programs but it may also raise environmental safety concerns. The environmental effects of genetically modified transgenic trees (GMTs) have been studied in many countries during the last 15 years. Today there is an urgent need of putting together this scattered knowledge to build-up a European knowledge platform for addressing GMTs in plantations. The main aims of Working Group 2 (WG2) of the European Cooperation in Science and Technology (COST) Action FP0905 “Biosafety of transgenic forest trees” (http://www.cost-action-fp0905.eu/) are (1) to discuss, based on scientific facts, whether current containment strategies are appropriate or need to be improved for GMTs, (2) to define a common protocol to track the transgene from the laboratory to the final product, and (3) to assess the possible impacts of GMTs on the environment. The potential risks of GMTs, the fate of recombinant material and the potential relevance of recombinant genes on plant´s omics are other main aspects to be considered and compared to similar processes with endogenous genes in conventional breeding. The group involves experts from public research, government and independent regulatory sectors across COST and non-COST member countries. The activities of this group have been organized into three Task Groups focusing on (1) risk assessment studies and guidance documents, (2) the monitoring of the transgenes and recombinant plant material, and (3) the impact of GMTs on exposed ecosystems. As a first step of WG2 activity, a database of guidance documents from national and transnational sources dealing with impacts and risk assessments of GMTs is being created to identify common and case-specific issues on biosafety. It is expected that the information gained will facilitate (1) a science-based understanding of the impacts of GMTs on the environment in comparison with that of traditionally tree breeding, and (2) future socio-economic and cost/benefits analyses of GMTs in plantations.

Simulated environmental criticalities affect transglutaminase of Malus and Corylus pollens having different allergenic potential

Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A(2) (sPLA(2)) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA(2) activity and putrescine conjugation to sPLA(2). We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.

Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

BackgroundThe molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro.MethodologyPlant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction.ConclusionsOur results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs.

Transgenic Tobacco Plants Resistant to Two Viruses via RNA Silencing

RNA silencing is a phenomenon of homologous RNA degradation induced by dsRNA, which is an effective strategy to obtain virus resistant plants so far. By means of this strategy, higher bio-security immune transgenic plants were obtained, avoiding recombining and transcapsidating with other virus genes. In this study, the recombinant plant expression vector pBIN438-MPRep (i/r) consisting the inverted repeat of TMV-ΔMP and CMV-ΔRep fusion fragment was transformed into tobacco cultivar K326 via Agrobacterium-mediated. The transformants were selected in the culture medium with 100 mg L^(-1) Kan. One hundred and ninety-six transgenic plants were obtained, one hundred and twenty-eight of which were positive plants, and the resistance to CMV and TMV was tested at the degree of virus. PCR-Southern blot and RT-PCR analysis of the transgenic plants demonstrated that the exogenous DNA was integrated into the tobacco genomic DNA and was expressed in transcriptional level. Resistance assay indicated that about 20.3% transgenic plants were immune to the co-infection with TMV and CMV. This result will provide to crops an important reference for plant anti-viral breeding and for preventing the viral co-infection.

Guanylyl cyclase activity in plants?

Cyclic nucleotides have defined signal transduction roles in animals, fungi, and other diverse eukaryote kingdoms. However, the situation in plants is still not clear after more than 3 decades of investigations reporting evidence for and against a regulatory role for cyclic nucleotides in higher plants. No sequenced higher plant genome contains homologs of the nucleotidyl cyclase genes that are recognizable in diverse eukaryotic kingdoms, including, curiously, the Chlorophyta phylum of the Viridiplantae kingdom. The report of Qi et al. (1) is a recent example of claims that recombinant plant proteins (or their fragments) from diverse protein families have guanylyl cyclase (GC) activity in vitro—claims that have led to the hope that the cyclic nucleotide regulatory system in plants is not a molecular house of cards. However, the GC activity reported was ≈150 fmol cGMP per μg of 14-kDa protein fragment (AtPepR1-GC) per 30 min. This activity is extraordinarily low. Each molecule of this putative recombinant GC synthesizes one cGMP molecule every 10 d. By contrast, recombinant and native human GC molecules synthesize 103–104 cGMP molecules per second, 109- to 1010-fold faster (2). This very low activity is typical of the other putative noncanonical GCs reported in plants, including fragments of other plant receptor kinases cited by Qi et al. (1) and Ludidi and Gehring (AtGC1, ref. 3). GC comprises 0.005–0.1% of bovine lung protein (2), but even if AtPepR1 was 99% of total Arabidopsis protein the GC activity of the plant would be <10−6 of the activity in some animal tissues.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Effective Removal of Bisphenol a from Contaminated Areas by Recombinant Plant Producing Lignin Peroxidase

We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. Lignin peroxidase is a well-known enzyme used for the degradation of some environmental pollutants. In the present study, we introduced an extracellular fungal enzyme, the lignin peroxidase of Trametes versicolor , into tobacco plants. Six transgenic plant, designated FLP-1, 2, 3, 4, 5 and 8, produced lignin peroxidase in a crude extract of the root. The FLP-1, FLP-2 and FLP-8 were able to remove 10μmol of bisphenol A g -1 dry weight from hydroponic culture. The efficiency of this removal was approximately 4-fold greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.

A kinetic comparison of asparagine synthetase isozymes from higher plants

Four previously identified maize asparagine synthetase (AsnS) genes and a soy AsnS gene have been cloned and expressed in Escherichia coli. The enzymes have been purified and kinetically characterized. The plant AsnS proteins were expressed mainly in the inclusion bodies although small amounts of one form (ZmAsnS2) were recovered in the soluble fraction. In order to measure the kinetic properties of these enzymes a sensitive assay based on the detection of Asn by HPLC has been developed. In addition a method to refold the recombinant plant AsnS to produce active enzyme has been developed. The plant AsnS enzymes are kinetically distinct with substantial differences in K(m) (Gln) and V(max) values when compared to each other. These differences may be important factors for transgenic studies using AsnS genes for crop improvement.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Phloridzin: Biosynthesis, distribution and physiological relevance in plants

The phenolic compound phloridzin (phloretin 2′- O-glucoside, phlorizin, phlorrhizin, phlorhizin or phlorizoside) is a prominent member of the chemical class of dihydrochalcones, which are phenylpropanoids. The apple tree ( Malus sp.) accumulates high amounts of phloridzin, whereas few other species contain this compound only in low amounts. Additionally, Malus sp. show a species- and tissue-specific distribution of phloridzin and its derivatives. Whereas the physiological role of phloridzin in planta is not fully understood, the effect on human health – especially diabetes – and membrane permeability is well documented. The biosynthesis of phloridzin was investigated only recently with recombinant enzymes and plant protein extracts and involved a NADPH-dependent dehydrogenase, chalcone synthase and UDP-glucose:phloretin 2′- O-glycosyltransferase.