An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.     

Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.     

Characterization of Isoforms of Pectin Methylesterase of Linum usitatissimum Using Polyclonal Antibodies

In flax (Linum usitatissimum, c.v. Ariane) pectin methylesterase(PME) (EC 3.1.1.11 [EC] ), ionically bound to cell-wall, was composedmainly of forms with isoelectric points (pIs) of 7.1, 7.6 and9.6. Minor forms, with acid pIs (4.5, 4.8 and 6.3), were detectedduring the purification of two of these forms. Polyclonal antibodieswere raised against the isoenzymes presenting pIs of 7.1 and7.6. Antibodies recognized antigenic forms and two close proteinsin the basic range which could be associated to the PME activitywith pI of 9.6. Antibodies did not recognize any acid formsand exhibited no cross-reactivity with proteins resolved inthe cellular content. Antigenicity was related mainly to theprotein part of the glycosylated enzyme. The antibodies againstflax PME did not cross-react with PMEs from Citrus and tomatoand with glycosylated proteins of various sources. Specificityof anti-PME antibodies was judged sufficient to localize therecognized forms on tissue prints of flax hypocotyls. AlthoughPME was distributed in the whole parts of hypocotyl, stainingwas not homogeneous and appeared reinforced in the apical zone.In the basal part, epidermis was more contrasted than internaltissues. (Received August 2, 1994; Accepted January 3, 1995)     

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a λgtll expression library constructed from MG pericarp poly(A)⁺ RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.     

Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.     

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.     

植物果胶甲酯酶及其花粉特异基因的分离

果胶甲酯酶与植物的多种重要生长发育过程有关,是目前植物生物学研究中的一个热点。根据相关文献,对植物果胶甲酯酶的结构模型、作用方式以及花粉特异的果胶甲酯酶基因的分离进行了综述。     

Purification and some properties of starch branching enzyme (Q-enzyme) from tuberous root of sweet potato

The pattern of isoforms of starch branching enzyme II or Q-enzyme II in the tuberous root of sweet potato was distinct from those of other organs; altogether 7 isoforms of QEII were contained in the sweet potato plant. The QEIIf isoform, one of the two major QEII isoforms in the tuberous root, was purified to homogeneity by using a variety of HPLC columns. The purified QEIIf was a monomeric protein with a molecular mass of about 85 kDa. Western blot analysis showed that the polyclonal antibodies raised against the purified QEIIf was significantly reactive to the rice endosperm QEI, but not to the rice endosperm QEIIa. Furthermore, the sweet potato QEIIf reacted with the antiserum raised against the rice endosperm QEI, but not with that against the rice endosperm QEIIa. The results suggest that the sweet potato QEIIf is more similar to the rice endosperm QEI than to the rice endosperm QEIIa.     

Pectin Methylesterase of Datura species,purification, and characterization from Datura stramoniumand its application

Pectin methylesterases (PME; EC 3.1.1.11) involved in de-esterification of pectin and have applicability in food, textiles, wines, pulp, and paper industries. In the present study, we compared PME activity of different parts of 3 Datura species and found that fruit coat showed maximum PME activity followed by leaf and seed. PME from leaves of D. stramonium (DsPME) was purified and characterized. DsPME showed optimum activity at 60 °C and pH 9 in the presence of 0.3 M NaCl. DsPME was stable at 70 °C and retained more than 40% activity after 60 min of incubation. However, enzyme activity completely abolished at 80 after 5 min of incubation. It follows Michaelis-Menten enzyme kinetics. Km and Vmax with citrus pectin were 0.008 mg/ml and 16.96 µmol/min, respectively. DsPME in combination with polygalactourenase (PGA) increased the clarity of orange, apple, pomegranate and pineapple juices by 2.9, 2.6, 2.3, and 3.6 fold, respectively in comparison to PGA alone. Due to very high de-esterification activity, easy denaturation and significant efficacy in incrementing clarification of fruit juice makes DsPME useful for industrial application.     

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrubThaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.     

Separation of pectin methylesterases and polygalacturonases on monolithic columns

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M(r) 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M(r) 82 kDa) and a basic protein with M(r) 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M(r) 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH(3)CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

烟草果胶甲基酯酶（PME）基因的克隆及功能分析(英)

为了研究果胶甲基酯酶（pectin methyl-esterase,PME）（EC 3.1.1.11）与植物病毒运动蛋白之间的相互作用,应用RT-PCR方法从烟草(Nicotiana benthamiana)中克隆了PME基因,并测定了全序列(GenBank登录号AY238968).序列分析显示该基因由两个保守的结构域组成(PMEI和pectinesterase). DNA印迹结果表明,该基因在基因组中存在多个拷贝,蛋白质印迹表明,植物总蛋白中存在两种形式的PME蛋白,但RNA印迹结果显示,在烟草细胞中只检测到全长的PME转录产物.酵母双杂交结果表明,PME与水稻矮缩病毒Pns11(具有非特异的核酸结合活性)之间存在相互作用,而没有检测到PME与已知的运动蛋白Pns6之间的相互作用,推测Pns11蛋白可能参与了水稻矮缩病毒粒子的运动.     

Identification of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation of fatty acid-derived flavor compounds

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.     

Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula x tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.     

Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit.

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.     

Comparative study of the inactivation kinetics of pectinmethylesterase in tomato juice and purified form

Pectinmethylesterase (PME) extracted from tomato fruit was purified by affinity chromatography. A single peak of PME activity was observed, presenting a molar mass of 33.6 kDa, an isoelectric point higher than 9.3, and an optimal temperature and pH for activity of 55 degrees C and 8.0, respectively. The processing stability of purified tomato PME in buffer solution was compared to PME stability in tomato juice. In both media, thermal inactivation of PME presented first-order inactivation kinetics, PME in tomato juice being more heat-labile than purified PME. Regarding high-pressure treatment, tomato PME showed to be very pressure-resistant, revealing an outspoken antagonistic effect of temperature and pressure. To avoid cloud loss in tomato juice, a time-temperature treatment of 1 min at 76.5 degrees C was calculated in order to have a residual PME activity of 1 x 10(-)(4) U/mL.     

Immunolocalization of a defense-related 87 kDa cystatin in leaf blade of tomato plants

In our previous work, we described the defensive potential of a wound- and methyl jasmonate-inducible 87 kDa tomato cystatin and its accumulation in a crystalline form. Here, we report the immunolocalization of this cysteine proteinase inhibitor in tomato leaf blade. Methyl jasmonate treated wild type plants showed accumulation of crystalline structures that were specifically and strongly stained with polyclonal antibodies against tomato cystatin. Crystalline cystatin was found in palisade and spongy parenchyma cells and immuno-gold electron microscopy analysis revealed that the crystals were compartmentalized in the cytosol. The same pattern in localization of cystatin was observed in transgenic tomato plants superexpressing prosystemin transgene. Our data showing the accumulation of cystatin in response to methyl jasmonate and in response to a overproduction of a wound signal corroborate the protective role of this inhibitor.