In Vitro and In Situ Properties of Cell Wall Pectinmethylesterases From Mung Bean Hypocotyls

Pectinmethylesterases (EC 3.1.1.11 [EC] ) have been solubilized fromyoung and mature tissues of mung bean hypocotyls. Whatever theplastic potential of the tissues, most of the pectinmethylesteraseactivity was located in the cell walls. Several active fractionswere obtained after chromatography on CM Sépharose. Equilibriumsedimentation in an analytical ultracentrifuge indicated theMW of the isolated isoforms to be close to 75 000 whereas SDS-PAGEelectrophoresis gave a MW around 32 000, suggesting the possibilityof dimeric structures. Mung bean pectinmethylesterase (PME)showed cross reactivity with soybean antiserum. Experiments carried out with p-nitrophenylacetate and Citruspectin revealed that PME and esterase activities might correspondto different isoforms. It was also noted that the stimulationinduced by cations was stronger when the enzymes were boundto the cell walls. The high ionic sensitivity suggested that,in situ, the ionic environment regulates pectinmethylesteraseactivity principally by modifying the pectin molecules, whichenhances the affinity of the enzymes for their substrate. Thesedata indicate the importance of the calcium content of the cellwalls and might explain the decrease in methylated pectins alongthe mung bean hypocotyl and, in turn, the loss of plasticity. Key words: Cell wall, hypocotyl, pectinmethylesterase, Vigna radiata     

Changes in pectinmethylesterase (PME) activity caused by ethylene applied at different temparatures

At 25?C the activity of pectinmethylesterase (PME) solutionincubated in ethylene atmosphere (100 µl/liter) was lowerthan that of PME solution incubated in air. On the other hand,at 6?C the activity of the ethylene-incubated PME was higherthan that of the air-incubated solution. The effects of bothtemparature and ethylene on PME-solution activity are reversiblephenomena. Ethylene accelerated both softening was acceleratedas PME activity in avocado fruit at 25?C. However at 6?C softeningwas accelerated as PME activity was promoted by ehtylene treatment.Ethylene had similar effects on PME activity in both purifiedenzyme solution and in vivo systems. (Received ; )     

Purification and Characterization of Pectinmethylesterase from Ficus awkeotsang Makino Achenes

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4°C for at least 6 months, and at 60°C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K_m value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V_max value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Novel role for pectin methylesterase in Arabidopsis: a new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

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.     

Immobilized and Free Apoplastic Pectinmethylesterases in Mung Bean Hypocotyl

The nature and the action pattern of apoplastic pectinmethylesterase (PME) isoforms were investigated in mung bean [Vigna radiata (L.) Wilzeck] hypocotyls. Successive extractions of neutral and alkaline PME isoforms present in hypocotyl native cell walls (referred to as PE1, PE2, PE3, PE4, with increasingly basic isoelectric points) revealed that solubilization of PE1, PE2, and PE4 did not induce any significant decrease in the cell-wall-bound PME activity. The in vitro de-esterification occurring when isolated cell walls were incubated with pectin resulted, then, from the activity of PE3. In addition, pH control of PME activity was shown to be much stronger for enzymes bound to cell walls, in their native state or reintroduced after solubilization, than for enzymes in solution. Mature cell walls showed much more activity than young cell walls, and were relatively enriched in two acidic PME isoforms missing in young cell walls. One acidic PME was also detected in the extracellular fluid. The acidic and neutral isoforms that could be easily transferred from their binding sites to their substrate might be those involved in the demethylation process developing along the mung bean hypocotyl.     

Immobilization of pectinmethylesterase from acerola (Malpighia glabra L.) in porous silica

The total and partially purified enzyme pectinmethylesterase from acerola fruit was covalently immobilized on porous silica particles. These efficiency values were 114% for the total PME and 351% for the partially purified PME. In both forms the immobilization resulted in compounds with high thermal stability.     

Action pattern of Valencia orange PME de-esterification of high methoxyl pectin and characterization of modified pectins

Valencia pectinmethylesterase (PME) fractions, B-PME, containing 36 and 13 kDa protein bands and U-PME, containing a 36 and 27 kDa protein bands, were used to de-esterify original pectin (O-Pec) from 73% degree of esterification (%DE) to 63% (B-Pec) and 61% DE (U-Pec), respectively. Most O-Pec eluted from ion exchange chromatography at low salt concentration and a smaller component eluted at higher ionic strength. B-Pec and U-Pec eluted as one broad peak at higher ionic strength. PME modification did not change molecular weight: O-pectin (134,000 g/mol), U-Pec (133,850 g/mol), and B-Pec (132,250 g/mol). The NMR signal of GG and GGG increased after modification, whereas the signal of EE and EEE decreased. The negative zeta-potential increased with pH for all pectins. U-PME and B-PME created differently modified pectins that vary in degree and length of multiple attacks and fraction of the pectin population that was modified.     

Cloning and immunolocalization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes

A 30-kDa protein extracted from the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes has been identified as a thermostable chitinase based on its enzymatic activity. A cDNA fragment encoding the precursor protein (including a cleavable signal sequence) of this chitinase was obtained by PCR cloning, and subsequently confirmed by immunological recognition of its overexpressed protein in Escherichia coli. Homology modeling predicted that this thermostable chitinase in jelly fig achenes comprised a stable (betaalpha)(8) barrel fold with three pairs of disulfide linkage. Immunostaining indicated that this chitinase was exclusively localized in the pericarpial region but not in the seed cells where bulky protein bodies and massive oil bodies were accumulated. Spore germination of Colletotrichum gloeosporioides, a common post-harvest pathogen infecting ripening fruit of jelly fig and many other fruits, was inhibited by this chitinase purified from achenes. It is suggested that the biological function of the thermostable chitinase in the pericarp of jelly fig achenes is to protect the nutritive seeds from fungal attack during fruit ripening.     

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)     

The Germination Characteristics of Tithonia rotundifolia

The achenes of the plant Tithonia rotundifolia show an embryodormancy, requiring a 3-month period of after-ripening to stimulategermination. Mechanical scarification did not enhance imbibitionof the achenes, indicating that the thick bi-layered achenewall does not impede water uptake. The light conditions necessaryfor optimal germination changed with achene age and the achenesbecame less sensitive to temperature. About 30–40% ofrecently harvested achenes germinated in the dark at 25 °C.Following a period of dry storage some of the achenes developeda light requirement and germination increased at 20, 25 and30 °C. Gibberellic acid stimulated the germination of achenestested after 12 weeks dry storage, and could substitute to someextent for the light and temperature requirements of the storedachenes. Tithonia rotundifolia, seed germination, gibberellic acid     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

Upwind Movement of Achenes of Centaurea eriophora L. on the Ground

The lightly compressed achenes of Centaurea eriophora L. beara pappus composed of stiff bristles at their apex and have anelaiosome appendage at their base. The pappus is ineffectivein keeping the achene wind-borne but does serve to regulatethe movement of the achene on the ground in response to wind.In wind the achene swivels like a weather vane with the baseof the achene pointing into the wind. In weak wind the pappusbristles prevent the achene from blowing away. In stronger windthe bristles move due to their flattened, flexible, hinge-likebases and act like ratchets against the substratum, thus enablingthe achene to move upwind. In either case achenes remain ingroups. Ants are attracted to the elaiosome and disperse theachenes. Wind-induced movement was explored by testing acheneson various substrata in a wind tunnel at free-stream speedsbetween 2 and 7 m s^-1. Wind dispersal; Centaurea eriophora ; seeds; achenes     

Germination of Tagetes minuta L. I. Temperature Effects

Initial studies have indicated that Tagetes minuta achenes haveboth a temperature and a light requirement for germination.Temperatures tested were 10, 20, 25, 30 and 35 °C. Germinationwas optimal at 25 °C under white light conditions. Underthese conditions 100 per cent of achenes germinated within 7days of imbibition. There was no germination at 10 or 35 °Ceither in the light or in the dark. Achenes imbibed and incubatedat 35 °C for 4 days showed no visible signs of germinationbut on transfer to 25 °C, 100 per cent of these achenesgerminated within 24 h. Furthermore, achenes given this hightemperature (35 °C) treatment could be dried at 25 °C,re-imbibed at 25 °C and again 100 per cent of achenes germinatedwithin 24 h of re-imbibition. This rapid germination responsefollowing removal from the high temperature regime could alsobe induced by transfer to temperatures of 20 °C or 20 °C(16 h) alternating with 10 °C (8 h). Tagetes minuta L., weed seeds, germination, temperature, light     

Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity

Purified ascorbate oxidase from Cucurbita pepo medullosa hasbeen subjected to enzymatic deglycosylation using peptide N-glycosidaseF. Experimental conditions were chosen to obtain efficientlydeglycosylated and active ascorbate oxidase: in particular,three different detergent solutions were added separately tothe incubation mixtures prior to the peptide N-glycosidase F.The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate+ 0.5% (v/v) Nonidet P-40 proved to be the only one effectivefor our purpose. Our results indicate that: (i) the presenceof detergents did not affect the enzymatic activity; (ii) fullydeglycosylated enzyme retained its activity compared with thenative form. Moreover, anti-native ascorbate oxidase antibodiesscarcely recognized deglycosylated protein. ascorbate oxidase blot deglycosylation     

Control of heat-shock response of young gametophytes of the sensitive fern is at the translational level

Young gametophytes of the sensitive fern, Onoclea sensibilis,respond to heat-shock by synthesizing in excess certain proteinsthat are made at normal growth temperature. Enhanced proteinsynthesis occurred during a 2 h heat-shock at a range of temperaturesbetween 38 °C and 50 °C. Although a temperature of 50°C proved lethal, a 5 min pulse at 50 °C resulted inenhanced synthesis of heat-shock proteins which continued forseveral hours at 25 °C. After heat-shock at 50 °C for10 or 15 min, the gametophytes temporarily lost their capacityfor protein synthesis but normal protein synthesis was resumedwithin 24 h of heat-shock. A heat-shock at 38 °C precedingone at 50 °C did not have any protecting effect on the gametophytes.In vitro translation of poly(A)+ RNA isolated from heat-shockedgametophytes yielded several proteins including heat-shock proteins.The results suggest that, rather than activating genes encodingnew messages for the synthesis of stress proteins, heat-shockof gametophytes of O. sensibilis triggers a controlling systemwhich enhances the translation of certain messages that aresynthesized at normal growth temperature. Key words: Onoclea sensibilis, heat-shock response, protein synthesis, sensitive fern, in vitro translation