The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor

Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME.     

Pectin methylesterase inhibitor cDNA from kiwi fruit

We have newly isolated one partial pectin methylesterase inhibitor (PMEI) and two full-length cDNA clones from a kiwi fruit cDNA library. The two full-length cDNA clones, Adpmei-1 and Adpmei-2, had an open reading frame of 185 amino acids, including a predicted signal peptide sequence necessary for localization in the cell-wall space. As the deduced amino acid sequence of the cloned fragment was almost same as the sequence of the previously purified PMEI protein (Camardella et al., Eur J Biochem 267:4561–4565), the clones were considered to be cDNAs encoding PMEI protein. Southern blot analysis indicated a low-copy number of the PMEI genes. Transgenic analysis of asparagus calli expressing a kiwi fruit PMEI gene driven by the CaMV 35S promoter demonstrated in vivo inhibition effects of PMEI on the endogenous pectin methylesterase (PME) activity. The relative expression levels of the PMEI genes in kiwi fruit, analyzed by competitive PCR, increased with the progression of fruit maturation. Given that PME activity also showed its highest level at the fully ripened stage of maturation, the increase in PMEI expression may not indicate direct inhibitory effects on the PME activity and fruit maturation process.     

Mago nashi Interacts with a Taiwania (Taiwania cryptomerioides) Pectin Methylesterase-like Protein

Mago nashi proteins are highly conserved among eukaryotes. They are involved in oogenesis, embryogenesis and germ-line determination during animal development, and play important roles in pollen tube growth, root development and spermatogenesis during plant development. In this study, we used yeast two-hybrid screening to show that the TcMago protein can interact with a Taiwania (Taiwania cryptomerioides) pectin methylesterase-like protein (TcPME1) which consists of a transmembrane domain, a pectin methylesterase inhibitor (PMEI) domain and a pectin methylesterase (PME) domain. The PME domain of TcPME1 was necessary for binding with the TcMago protein. The PME domain was highly conserved in all the plants assayed and had five well conserved active site residues. The predicted protein tertiary structures revealed that the PMEI domain and PME domain of TcPME1 are similar to kiwi (Actinidia deliciosa) PMEI and carrot (Daucus carota) PME, respectively. TcPME1 was expressed abundantly in the early stage of root elongation and accumulated at root tip. Moreover, TcPME1 expression was inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Thus, TcPME1 might be involved in root elongation, shoot development and auxin transport during Taiwania development.     

Pectin methylesterase inhibitor

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.     

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.     

Kiwi fruit PMEI inhibits PME activity,modulates root elongation and induces pollen tube burst in Arabidopsis thaliana

Pectins are major components of primary cell wall that play a crucial role in plant development. After biosynthesis, pectins are secreted in the cell wall by Golgi-derived vesicles under a highly methylesterified form and are de-methylesterified by pectin methylesterases (PME). It is hypothesized that PME might be regulated by pectin methylesterase inhibitor (PMEI). In this paper, we show by isoelectric focalisation and subsequent zymogram that kiwi PMEI was able to inhibit Arabidopsis PME activity by forming a complex. The complexes were stable under a wide range of ionic strength and pH. Moreover, PMEI might be able to form a complex with basic PMEs including three PMEs strongly expressed in root and four PMEs expressed in pollen grains. Finally, exogenous treatment with kiwi PMEI was able to reduce the activity of cell wall resident PMEs with persistent effects such as an increase of the root growth and a dramatic effect on pollen tube stability.     

Nutritional and gibberellic acid requirements in kiwifruit vitroponic cultures

Summary Residual macronutrients, carbon source, and gibberellic acid (GA₃) in the culture medium were measured throughout the whole culture period of kiwi (Actinidia deliciosa Chev. cv. Hayward) explants cultured in liquid medium using cellulose plugs as explant support. The objective of this study was to adapt the composition of the culture medium to an automated culture system with the possibility of applying 6-benzyladenine pulses so as to improve the efficiency of kiwifruit micropropagation. Ammonium and phosphate were the most consumed ions at the end of the culture period (35 d). Murashige and Skoog medium might be too rich for the culture of kiwifruit in liquid medium except with respect to phosphate which decreased significantly toward the end of the culture period. Sucrose, and the glucose resulting from the hydrolysis of the former, were taken up by the kiwi explants throughout the whole culture period. GA₃ absorption by the kiwi explants occurred gradually throughout the subculture, although 67% of this initially added plant growth regulator remained in the culture medium at the end of the 35-d culture period.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

Kiwellin, a Novel Protein from Kiwi Fruit. Purification, Biochemical Characterization and Identification as an Allergen*

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit. *The protein sequence data reported in this paper will appear in the Swiss-Prot and TrEMBL knowledgebase under the accessionnumber P84527.     

1-Methylcyclopropene and ethylene as regulators of in vitro organogenesis in kiwi explants

In this paper, we study the influence of ACC, AVG and 1-MCP on in vitro organogenesis of kiwi (Actinidia deliciosa) explants and on ethylene metabolism. Results indicated that increasing ethylene production and accumulation in the head space of the culture vessel by adding ACC to the culture medium inhibited organogenesis, except for rooting, which increased and stimulated ACC oxidase activity threefold, whereas AVG increased the length and number of shoots and leaves and inhibited about 80% ACC synthase activity compared with the reference explants. 1-MCP exerts a stimulatory effect analogous to AVG, increasing ACC synthase activity and organogenesis of kiwi explants. This effect is not reverted by the addition of ACC to the culture medium. Therefore, ethylene production and its accumulation in the headspace of the culture vessels seems to be responsible for the inhibition of shoot development as well as increasing rooting in in vitro cultured kiwi explants.     

Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.     

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.     

Terpene glucoside production: Improved biocatalytic processes using glycosyltransferases

Terpenoids are one of the main classes of natural products. In plants, a large fraction of the terpenoids is present as nonvolatile glycosides. The terpene glycosides have attracted much attention as antimicrobials, flavor precursors, and detergents. They are either extracted from plant materials or are synthesized by chemical and biocatalytic methods. Up to now, biotechnological production of terpene glycosides is based on reversed hydrolysis performed by glycosidases. However, this method suffers from low yields as a matter of principle. Recently, the first uridine diphosphate‐glucose:monoterpenol β‐d ‐glucosyltransferase (GT) genes were cloned and characterized from grapevine (Vitis vinifera) and kiwi (Actinidia deliciosa). Heterologous expression in Escherichia coli yielded promiscuous GT enzymes that efficiently glucosylated primary monoterpenols, simple alcohols, and phenols. The GT enzymes differed in substrate preference and activity toward their terpenoid substrates. Biotransformation experiments confirmed the applicability of the novel GTs in biocatalytic processes for the production of these novel compounds. In the near future, terpene glucosides will become commercially available for food, cosmetic, and pharmaceutical industry due to improved biocatalytic processes involving GT enzymes.     

Xylem Sap from Actinidia chinensis: Apparent Differences in Sap Composition Arising from the Method of Collection

Large differences in composition were found between xylem sapcollected from Actinidia chinensis (Chinese gooseberry or kiwifruit) as bleeding sap and sap collected by vacuum extraction.A comparison of saps collected by the two methods has littlemeaning, however, unless the position on the plant from whichsap was collected and the prior treatment of the plant are specified.Furthermore the composition of bleeding sap changes rapidlywith time, probably because of marked gradients in concentrationof individual solutes in the xylem sap from the base to thetop of the plant. Contamination of vacuum-extracted sap by cellularcontents was shown to be insignificant. Sap collected as bleeding sap and by vacuum extraction are ofsomewhat different origins. It would be difficult to predictthe composition of bleeding sap simply from a knowledge of vacuum-extractedsap: it may be similarly unwise to predict the composition ofthe transpiration stream from that of vacuum extracted sap. Actinidia chinensis, kiwi fruit, Chinese gooseberry, xylem sap, bleeding sap, vacuum-extraction     

Influence of Cultivation Parameters on the Mineral Composition of Kiwi Fruit from Corsica

The effect of four cultivation parameters (postmaturity harvest date, storage period at 0 °C, and input of nitrogen and potassium fertilizers) on the mineral composition of kiwi fruit (Actinidia deliciosa var. H ayward) from Corsica were evaluated. The kiwi fruit were harvested on three dates at two‐week intervals and some fruit were stored for three and four months. The kiwi fruit orchard was fertilized with controlled levels of nitrogen (five levels) and potassium (three levels) during one growing season. The concentrations of 67 elements in kiwi fruit were measured using various analytical methods, such as flow injection spectrophotometry, flame atomic absorption spectrometry, flame atomic emission spectrometry, electrothermal atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry, and filtration. The main elements in kiwi fruit are K, N, Cl, P, and Si and, to a lesser amount, Ca, Mg, Na, and Fe. This study demonstrates a high degree of difference in the amount of 23 mineral elements depending on the harvest date, the time of storage, and the input of fertilizers.     

Identification and Quantification of Ascorbic Acid in Kiwi Fruit by High-Performance Liquid Chromatography

The content of ascorbic acid in kiwi fruit (Actinidia chinensisPlanch) of various cultivars was determined by high-performance liquid chromatography (HPLC). A minimal content of ascorbic acid was found in fruits of Gaivard cultivar: in juice – 5.44, skin – 1.14, and pulp – 4.20 mg/g.     

Amplification,sequencing and characterization of pectin methyl esterase inhibitor 51 gene in Tectona grandis L.f.

Tectona grandis L.f. (Teak), a very important source of incomparable timber, withstands a wide range of tropical deciduous conditions. We achieved partial amplification of pectin methylesterase inhibitor 51 (PMEI) gene in teak by E. pilularis cinnamoyl Co-A reductase (CCR) gene specific primer. The amplified teak gene was of 750 bp, 79% identity and 97% query cover with PMEI of Sesamum indicum. The phylogenetic tree clustered the amplified gene with PMEI of database plant species, Erythranthe guttata and Sesamum indicum (87% bootstrap value). On conversion to amino acid sequence, the obtained protein comprised 237 amino acids. However, PMEI region spanned from 24 to 171 amino acids, 15.94 kDa molecular weight, 8.97 pI value and C₆₉₇H₁₁₁₇N₁₉₉O₂₁₁S₉ molecular formula with four conserved cysteine residues as disulfide bridges. 25.9 % protein residues were hydrophilic, 42.7% hydrophobic and 31.2% neutral. Teak 3D PMEI protein structure corresponded well with Arabidopsis thaliana and Actinidia deliciosa PMEIs. The gene maintains integrity of pectin component of middle lamella of primary cell wall and confers tolerance against various kinds of stresses. Teak conferred with overexpression of PMEI may secure a wide adaptability as well as luxuriant timber productivity and quality in adverse/ fluctuating/ scarce climatic and environmental conditions of tropical forests.     

A Comparative Genome Analysis of PME and PMEI Families Reveals the Evolution of Pectin Metabolism in Plant Cell Walls

Pectins are fundamental polysaccharides in the plant primary cell wall. Pectins are synthesized and secreted to cell walls as highly methyl-esterified polymers and then demethyl-esterified by pectin methylesterases (PMEs), which are spatially regulated by pectin methylesterase inhibitors (PMEIs). Although PME and PMEI genes are pivotal in plant cell wall formation, few studies have focused on the evolutionary patterns of the PME and PMEI gene families. In this study, the gene origin, evolution, and expression diversity of these two families were systematically analyzed using 11 representative species, including algae, bryophytes, lycophytes and flowering land plants. The results show that 1) for the two subfamilies (PME and proPME) of PME, the origin of the PME subfamily is consistent with the appearance of pectins in early charophyte cell walls, 2) Whole genome duplication (WGD) and tandem duplication contribute to the expansion of proPME and PMEI families in land plants, 3) Evidence of selection pressure shows that the proPME and PMEI families have rapidly evolved, particularly the PMEI family in vascular plants, and 4) Comparative expression profile analysis of the two families indicates that the eudicot Arabidopsis and monocot rice have different expression patterns. In addition, the gene structure and sequence analyses show that the origin of the PMEI domain may be derived from the neofunctionalization of the pro domain after WGD. This study will advance the evolutionary understanding of the PME and PMEI families and plant cell wall development.