Pectinolytic enzymes associated with the soft rots ofCitrus sinensis caused byAspergillus aculeatus andBotryodiplodia theobromae

Botryodiplodia theobromae andAspergillus aculeatus causing soft rots ofCitrus fruits were grown on wheat offal medium. The filtered washings of the wheat offal cultures were then assayed and found to contain pectinmethylesterases (PME) and polygalacturonases (PG). The two pectinases were also detected in large quantities from the filtrates of rotted fruits ofCitrus sinensis incited by each of the two test fungi. Two enzyme filtrates, one from rotted fruits ofCitrus sinensis and the other made up of pectinases from fungal cultures were introduced onto sterilised degreened fruits. Each of the two filtrates induced rot symptoms similar to those incited by the test fungi.Aspergillus aculeatus produced less PME and more PG thanBotryodiplodia theobromae.     

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.     

Postharvest Variation in Cell Wall-Degrading Enzymes of Papaya (Carica papaya L.) during Fruit Ripening

Pectin methylesterase (PME), polygalacturonase (PG), xylanase, cellulase, and proteinase activity were determined and related to respiration, ethylene evolution, and changes in skin color of papaya (Carica papaya L.) fruit from harvest through to the start of fruit breakdown. PME gradually increased from the start of the climacteric rise reaching a peak 2 days after the respiratory peak. PG and xylanase were not detectable in the preclimacteric stage but increased during the climacteric: during the post climacteric stage, the PG declined to a level one-quarter of peak activity with xylanase activity returning to zero. Cellulase activity gradually increased 3-fold after harvest to peak at the same time as PME, 2 days after the edible stage. Proteinase declined throughout the climacteric and postclimacteric phases. A close relationship exists between PG and xylanase and the rise in respiration, ethylene evolution, and softening. Cultivar differences in postclimacteric levels of enzymic activity were not detected.

An inhibitor of cellulase activity was detected in preclimacteric fruit. The inhibitor was not benzyl isothiocyanate (BITC). BITC did inhibit PG activity, though no inhibitor of PG activity was detected in preclimacteric homogenates when BITC was highest. The results indicate that inhibitors did not play a direct role in controlling wall softening.

     

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.This study was supported by grant No. 522/02/0708 of the Grant Agency of the Czech Republic.     

Tomato Fruit Polygalacturonase Isozyme 1 (Characterization of the [beta] Subunit and Its State of Assembly in Vivo)

Polygalacturonase isozyme 1 (PG1) is a heterodimer comprising a catalytic and noncatalytic or [beta] subunit, whereas polygalacturonase isozyme 2 (PG2) comprises only the catalytic subunit. To assess the state of assembly of PG1 in vivo, both subunits were purified to homogeneity and used to study assembly of the heterodimer. PG1 could be reconstituted in vitro from purified [beta] subunit and purified PG2 under a wide range of salt and pH conditions, and PG1 reconstituted in vitro was indistinguishable from PG1 isolated from tomato (Lycopersicon esculentum) fruit. Specific antibodies indicated that the [beta] subunit was present in fruit of all developmental stages, but absent in vegetative tissue. The state of assembly of PG1 in vivo was tested based on the differential thermal stability of PG1 and PG2 by heating segments of ripe fruit pericarp tissue. Temperatures well below those required to inactivate PG1 in vitro caused the loss of activity of both PG1 and PG2, suggesting that only heat-labile PG2 is present in vivo. In addition, when extracts of ripe fruit were rigorously maintained and analyzed at 4[deg]C, PG1 was absent or barely detectable. These results are consistent with the hypothesis that PG1 can assemble spontaneously and is essentially absent in intact tomato fruit but forms artifactually from PG2 and the [beta] subunit during the extraction of tomato fruit tissue when low temperatures are not rigorously maintained.     

Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal)

In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO₃, NaNO₃, and CaCl₂. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO₃ compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO_3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl₂ produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.     

Flipping the cyclooxygenase (Ptgs) genes reveals isoform-specific compensatory functions,

Two prostaglandin (PG) H synthases encoded by Ptgs genes, colloquially known as cyclooxygenase (COX)-1 and COX-2, catalyze the formation of PG endoperoxide H₂, the precursor of the major prostanoids. To address the functional interchangeability of these two isoforms and their distinct roles, we have generated COX-2>COX-1 mice whereby Ptgs2 is knocked in to the Ptgs1 locus. We then “flipped” Ptgs genes to successfully create the Reversa mouse strain, where knock-in COX-2 is expressed constitutively and knock-in COX-1 is lipopolysaccharide (LPS) inducible. In macrophages, flipping the two Ptgs genes has no obvious impact on COX protein subcellular localization. COX-1 was shown to compensate for PG synthesis at high concentrations of substrate, whereas elevated LPS-induced PG production was only observed for cells expressing endogenous COX-2. Differential tissue-specific patterns of expression of the knock-in proteins were evident. Thus, platelets from COX-2>COX-1 and Reversa mice failed to express knock-in COX-2 and, therefore, thromboxane (Tx) production in vitro and urinary Tx metabolite formation in COX-2>COX-1 and Reversa mice in vivo were substantially decreased relative to WT and COX-1>COX-2 mice. Manipulation of COXs revealed isoform-specific compensatory functions and variable degrees of interchangeability for PG biosynthesis in cells/tissues.     

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.     

Inhibition of o(2) consumption resistant to cyanide and its development by N-propyl gallate and salicylhydroxamic Acid

Kinetics of inhibition of cyanide-insensitive O₂ uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum `Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K_I for SHAM was similar in mitochondria and slices. However, the apparent K_I for PG in mitochondria was about 40-fold lower than the K_I for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway.     

Influence of various nitrogen and carbon sources on the production of pectolytic,cellulolytic and proteolytic enzymes byAspergillus niger

Three isolates ofAspergillus niger produced polygalacturonase (PG) and pectin methyl galacturonase (PMG) in the presence of organic and inorganic nitrogen sources. Complete inhibition of PG PMG cellulase (C_x) and proteinase synthesis was found in the presence of cystine in all isolates. Maximum biomass was found in sodium nitrate whereas no isolate could grow in the presence of cystine. A correlation between biomass and enzyme production could be obtained when sodium nitrate and cystine were added to the medium separately. All isolates produced pectic cellulolytic and proteolytic enzymes in the presence of various native carbon sources. Sodium polypectate was found to be the best carbon source for the production of PG and PMG; pectin inhibited completely the production of PG and PMG. Maximum cellulase production was brought about by cotton in all three isolates. Maximum proteinase production was observed with gelatin which served as poor substrate for fungal growth. Sucrose supported maximum fungal growth in comparison with all other native carbon sources. The increased production of pectolytic cellulolytic and proteolytic enzymes in the presence of sodium polypectate reflected a stimulation rather than an induction of synthesis of these enzymes.     

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.     

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of⁷⁵Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of⁷⁵Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.     

Changes in phospholipid composition of Thiobacillus neapolitanus during growth

Summary During the stationary growth phase, the phospholipids of Thiobacillus neapolitanus consisted of phosphatidyl glycerol (PG), diphosphatidyl glycerol (DPG), phosphatidyl-N-monomethylethanolamine (PME) and phosphatidyl ethanolamine (PE) in increasing amounts. In general, the phospholipids increased to a maximum concentration during the stationary phase and then decreased in concentration. Individually, PG and PE increased to a maximum in late lag or early exponential phase and then decreased in concentration. DPG and PME increased during the transition between the exponential and the stationary phase and reached a maximum concentration in the stationary phase. In older cultures, a quantitative interconversion between PG and DPG and PE and PME was observed. A lyso-phospholipid compound also appeared in the late stationary phase.The phospholipid composition of the culture supernatant fluid was essentially similar to that of the cells at all stages of growth. No excessive secretion of these products into the medium was observed at any growth stage of the culture.Abbreviations used PG Phosphatidyl glycerol - DPG Diphosphatidyl glycerol - PME Phosphatidyl-N-monomethylethanolamine - PE Phosphatidyl ethanolamine - GPGPG Glycerophosphoryl glycerophosphoryl glycerol - GPG Glycerophosphoryl glycerol - GPE Glycerophosphoryl ethanolamine - GPME Glycerophosphoryl-N-monomethylethanolamine     

Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum

Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl₂‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.     

How the rice weevil breaks down the pectin network: Enzymatic synergism and sub-functionalization

Pectin is the most complex polysaccharide in nature and highly abundant in plant cell walls and middle lamellae, where it functions in plant growth and development. Phytopathogens utilize plant pectin as an energy source through enzyme-mediated degradation. These pectolytic enzymes include polygalacturonases (PGs) of the GH28 family and pectin methylesterases (PMEs) of the CE8 family. Recently, PGs were also identified in herbivorous insects of the distantly related plant bug, stick insect and Phytophaga beetle lineages. Unlike all other insects, weevils possess PMEs in addition to PGs. To investigate pectin digestion in insects and the role of PMEs in weevils, all PME and PG family members of the rice weevil Sitophilus oryzae were heterologously expressed and functionally characterized. Enzymatically active and inactive PG and PME family members were identified. The loss of activity can be explained by a lack of substrate binding correlating with substitutions of functionally important amino acid residues. We found subfunctionalization in both enzyme families, supported by expression pattern and substrate specificities as well as evidence for synergistic pectin breakdown. Our data suggest that the rice weevil might be able to use pectin as an energy source, and illustrates the potential of both PG and PME enzyme families to functionally diversify after horizontal gene transfer.     

Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution

Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia inflata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauliflower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not differ in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these trangenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modifications of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed significant differences in their cation-binding properties in comparison with the wild-type. In particular, cell walls displayed increased affinity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modified. Indications of PME-mediated differences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no effects on the chemical structure of pectin from tuber cell walls could be detected. Received: 24 March 1999 / Accepted: 20 August 1999     

Production of pectolytic enzymes fromErwinia grown on different carbon sources

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.     

Plasma Membrane Alterations in Callus Tissues of Tuber-bearing Solanum Species during Cold Acclimation

Plasma membrane alterations in two tuber-bearing potato species during a 20-day cold acclimation period were investigated. Leaf-callus tissues of the frost-resistant Solanum acaule Hawkes `Oka 3878' and the frost-susceptible, commonly grown Solanum tuberosum `Red Pontiac,' were used. The former is a species that can be hardened after subjecting to the low temperature, and the latter does not harden. Samples for the electron microscopy were prepared from callus cultures after hardening at 2 C in the dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acaule increased in frost hardiness from −6 to − 9 C (killing temperature), whereas frost hardiness of S. tuberosum remained unchanged (killed at −3 C). Actually, after 15 days acclimation, a −9 C frost hardiness level in S. acaule callus cultures had been achieved.