Pectin Methylesterase

Fruit ripening is characterized by progressive depolymerisation of pectic and hemicellulosic polysaccharides of the cell wall. Several cell-wall hydrolases have been studied and their role implicated in the softening of various fruits. However, studies related to pectate lyases (PL), which degrade pectins through b-elimination of glycosidic linkages, have been very limited in fruit tissue. We have cloned a PL homologue, MiPel1 from ripening mango (Mangifera indica var. Dashehari), which shows sequence similarity to higher plant PL genes. The 1.7-kb complementary DNA of MiPel1 (accession number AY987389) encodes a putative protein of 437 amino acids, which possesses all the motifs and active site residues conserved in other PL. A progressive increase in transcript accumulation was observed during ripening but expression was delayed significantly in 1-methylcyclopropene-treated and control fruits kept in air without exogenous ethylene. The expression was specific to fruits and triggered only during ripening. No transcript was detected in other tissues such as leaf, stem and flower and during development of the fruit. The increase in transcript accumulation of MiPel1 during ripening was associated with an increase in the Ca 12 -dependent PL activity and pectin solubilization. It is proposed that expression of PL may be closely associated with pectin degradation during ripening and play an important role in mango softening.

Ohmic heating (OH) which is among to electrothermal methods and helps to inactivate microorganisms and enzymes was used in this study as thermal treatment on orange juice production for pectin methylesterase (PME) inactivation. Response surface methodology (RSM) was used for optimization of OH conditions. The effects of voltage gradient and temperature (independent variables) were investigated on PME activity (response) of orange juice. After optimization orange juice was produced and compared with untreated control juices and conventional thermally heated juices on the aspect of PME inactivation and some quality characteristics. Reduction of PME activities was found approximately 96 % in OH groups where conventional thermally heated juice has 88.3 % reduction value. Total pectin content was increased 1.72-2 % after OH applications. Ascorbic acid contents of OH samples were found between 43.08-45.20 mg/100 mL where conventional thermally heated juice has 42.9 mg/100 mL. As a result, it was determined that OH can be applied as a thermal treatment on orange juice production in moderate temperatures for PME inactivation and may improve functional properties of orange juice.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular ...

Mature, healthy green orange fruits inoculated with the spore suspension of Lasiodiplodia sp.IMI 503248 were immersed in hot water from 35 to 55C and varying exposure times from 5 to 30minutes, and incubated for ten days at 28+2C and 95 – 100% relative humidity inside sterilized glass desiccators. After incubation, cellulase, Pectin Methylesterase (PME) and Polygalacturonase (PG) activities were assayed in the different temperature time combinations in partially purified extracts by Ammonium sulphate precipitation. Healthy and untreated infected fruits served as controls 1 and 2 respectively. Investigation revealed that healthy (Control 1) and orange fruits treated at 35C for 25 and 30minutes showed no detectable cellulase activity comparatively with untreated infected (Control 2) and fruits treated above 35C that exhibited detectable cellulase activity. The set of fruits with no cellulase activity remained green with no disease symptom by 10 th day of storage while those with cellu...

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/ degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

The enzyme pectin methylesterase (PME) inactivation in orange juice was studied by both conventional and microwave heat treatment in batch conditions. Conventional heat treatment was carried out in a thermostatic bath, maintaining the temperature constant during the process. Different temperatures were tested at the interval between (40 and 95) °C and time between (0 and 60) s. The microwaveassisted heating process took place through two equipments: the first one have used a multi-mode microwave digestor, and the second a chemical and enzymatic synthesis mono-mode microwave reactor. Temperatures have been tested in the range of (40 to 90) °C and retention time between (0 and 60) s. The temperature's history has been acquired in real time for all processes. Physical-chemical characterization of the non-processed juice, pectin methylesterase enzyme activity, acidity and soluble solids content were conducted for both, fresh and treated juice. The results demonstrated that the microwave-assisted pasteurization was more effective than conventional, reaching 90 % inactivation at 60 °C after 10 s and reaching 99 % inactivation when exposed to 80 °C by at least 20 s.

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22°C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0°C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57°C) and a biphasic model for higher temperatures (58-70°C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.

The control of gynoecium development in Arabidopsis thaliana by the auxin response factor ETTIN (ETT) correlates with a reduction in the methylesterification of cell-wall pectins and a decrease in cell-wall stiffness in the valve tissues of the ovary. Here, we determine the list of genes rapidly regulated following the in-vivo activation of an ETT fusion protein, and show these to be significantly enriched in genes encoding cell-wall proteins, including several pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs). We also perform a genome-wide scan for potential ETT-binding sites, and incorporate the results of this procedure into a comparison of datasets, derived using four distinct methods, to identify genes regulated directly or indirectly by ETT. We conclude from our combined analyses that PMEIs are likely to be key actors that mediate the regulation of gynoecium development by ETT, while ETT may simultaneously regulate PMEs to prevent exaggerated developmental effects from the regulation of PMEIs. We also postulate the existence of one or more rapidly-acting intermediate factors in the transcriptional regulation of PMEs and PMEIs by ETT.

Effect of treatment of hot break single strength tomato juice (SSTJ) with different concentrations of exogenous pectin methylesterase (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 U/ 100 ml tomato juice) (PME) produced by Aspergillus niger under optimum conditions (45°C and  pH= 4  for 10 min in a water bath) selected based on a preliminary experiment was investigated in this study. The maximum viscosity value (342 cP) for tomato juice was obtained when pectin methylesterase concentration was added as  (25 U/ 100 ml juice). The changes in some properties of (SSTJ) were evaluated after pasteurization and bottling in amber glass bottles and during storage at 4 and 22°C for 6 weeks. It was found that there was a significant increasing in viscosity for enzymatic treated (SSTJ) samples after pasteurization, comparatively to the viscosity value of enzymatic untreated SSTJ (control), storage for 6 weeks at two respective temperatures, caused an insignificant decreasing in the viscosity of e...

Effect of treatment of hot break single strength tomato juice (SSTJ) with different concentrations of exogenous pectin methylesterase (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 U/ 100 ml tomato juice) (PME) produced by Aspergillus niger under optimum conditions (45°C and pH= 4 for 10 min in a water bath) selected based on a preliminary experiment was investigated in this study. The maximum viscosity value (342 cP) for tomato juice was obtained when pectin methylesterase concentration was added as (25 U/ 100 ml juice). The changes in some properties of (SSTJ) were evaluated after pasteurization and bottling in amber glass bottles and during storage at 4 and 22°C for 6 weeks. It was found that there was a significant increasing in viscosity for enzymatic treated (SSTJ) samples after pasteurization, comparatively to the viscosity value of enzymatic untreated SSTJ (control), storage for 6 weeks at two respective temperatures, caused an insignificant decreasing in the viscosity of enzymatic treated (SSTJ) samples. A significant increasing in GalA for WSP was observed in juices after their pasteurization and storage at 4 and 22°C for 6 weeks. The results indicated that there were significant decreasing in GalA for CSP whereas, the decreasing of GalA for NSP was insignificant for enzymatic treated tomato juices as compared with untreated tomato juices. The DM for WSP and CSP in treated samples were significantly lower than that for untreated samples after pasteurization and bottling and during storage for 6 weeks at 4 and 22°C. The DM of NSP could not be assaying due to the saponification of methylesters during the alkali extraction of this fraction .

Biochemical and physical changes in fruits of four guava cultivars during growth and development were studied. Investigation showed that protein content decreased markedly with fruit growth and development for all cultivars. The maximum level of reduction varied from 0.25 to 0.27%. Moisture content was significantly increased with fruit growth and development in all cultivars. The maximum level varied from 6.2 to 76.0%. Alcohol-insoluble solids (AIS) declined gradually during the initial growing period followed by rapid decline during later stages of development for all cultivars, which differed in their final value (8,67-13.33%). Water-soluble pectin for all cultivars increased gradually with fruit development. The maximum level varied from 0.34 to 0.64%. For all cultivars texture declined gradually during fruit development. The skin colour of the fruit changed gradually from dark green to yellow for all cultivars. Fruits picked before day 106 after fruit set had a reading of more than 30 psi (1 psi = 7.9 kPa) which was outside the range of the pressure tester, and thereafter pressure declined rapidly for all cultivars. Fruit volume increased rapidly with fruit development for all cultivars. Softness and yellowness of fruit were associated with lower protein and AIS contents, higher moisture and appreciable amounts of water-soluble pectin

Biochemical and physical changes in fruits of four guava cultivars during growth and development were studied. Investigation showed that protein content decreased markedly with fruit growth and development for all cultivars. The maximum level of reduction varied from 0.25 to 0.27%. Moisture content was significantly increased with fruit growth and development in all cultivars. The maximum level varied from 6.2 to 76.0%. Alcohol-insoluble solids (AIS) declined gradually during the initial growing period followed by rapid decline during later stages of development for all cultivars, which differed in their final value (8,67-13.33%). Water-soluble pectin for all cultivars increased gradually with fruit development. The maximum level varied from 0.34 to 0.64%. For all cultivars texture declined gradually during fruit development. The skin colour of the fruit changed gradually from dark green to yellow for all cultivars. Fruits picked before day 106 after fruit set had a reading of more than 30 psi (1 psi = 7.9 kPa) which was outside the range of the pressure tester, and thereafter pressure declined rapidly for all cultivars. Fruit volume increased rapidly with fruit development for all cultivars. Softness and yellowness of fruit were associated with lower protein and AIS contents, higher moisture and appreciable amounts of water-soluble pectin

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22°C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0°C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57°C) and a biphasic model for higher temperatures (58-70°C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.

This study evaluated the residual activities of amyloglucosidase (AMG) at 65 and 80°C, glucose oxidase (GO) at 50 and 75°C and neutral protease at 20 and 55°C after 3 passes of high pressure homogenization (HPH) at 150 MPa and 200 MPa (neutral protease and AMG) and at 100 and 150 MPa (GO). The results for AMG and neutral protease showed that the improvement in maximum enzyme activity was reached after one pass at 200 MPa, with an increment in the AMG residual activity measured at 80°C (activity increased from 13% to 21%) and in the neutral protease residual activity at 20°C (activity increased from 50% to 64%). However, the multiple passes caused no improvement in the activities of the enzymes. To the contrary, the results obtained for GO showed that HPH at 150 MPa continuously improved the activity at 75°C up to three passes, reaching an activity three times higher than the native sample. Additionally, it was observed that two passes of GO at 100 MPa resulted in the same level of GO activation reached after a single pass at 150 MPa. These results suggest that multiple HPH effects differ for each enzyme evaluated and can be applied to improve GO activity. Industrial relevance: Enzymes can be used in several areas of food industry. However, the price of enzymes and their low stability limits the enzymes' application in food industry. The HPH is suggested to process the enzyme prior to application, to increase enzyme activity mainly at non-ideal temperature. Therefore, the application of high pressure homogenization to improve enzymes activity can expand the range of enzyme uses.

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.

Pectins are fundamental polysaccharides in the plant primary cell wall. Polygalacturonases (PGs) and pectin methylesterases (PMEs), major components of the pectin remodeling and disassembly network, are involved in cell separation processes during many stages of plant development. A comprehensive study of these genes in plants could shed light on the evolution patterns of their structural development. In this study, we conducted whole-genome annotation, molecular evolution and gene expression analyses of PGs and PMEs in Brassica rapa and 8 other plant species. A total of 100 PGs and 110 PMEs were identified in B. rapa; they primarily diverged from 12-18 MYA and PMEs were retained more than PGs. Along with another 305 PGs and 348 PMEs in the 8 species, two different expansion or evolution types were discovered: a new branch of class A PGs appeared after the split of gymnosperms and angiosperms, which led to the rapid expansion of PGs; the pro domain was obtained or lost in the proPME...

Pectin is a very important ingredient in food processing. The present study was designed to find out the changes taken place in pectin content during development and ripening phases of banana fruit. The expression of gene (pectin esterase) was also observed during development and ripening. Five exotic cultivars namely William, Brazilian, G-Naine, Basrai and Pisang were selected for this study. Sampling were carried out after every 2 weeks during development and there was 6 sampling stages (from emergence to harvesting stage). After harvesting the fruit of all cultivars were subjected to natural ripening in the lab and sampling during ripening were carried out according to the colour changes in the fruit skin. 5 ripening stages were studied. Hence total number of 11 stages were studied during development and ripening. The sequence of pectin esterase were obtained from NCBI, primers were designed and procured. RNA was extracted from each cultivar and expression analysis of pectin esterase was carried out. It was found that the expression of gene is variable during the development and the ripening stages in each cultivar. Similarly, the pectin content were also significantly variable during development and ripening of fruit. The variation was also found among the cultivars. It was concluded that unripe banana have more pectin content then ripe banana and the pectin content of the fruit is associated with expression of the gene.

Experiments were conducted to examine softening and quality responses of harvested banana fruit to cold shock treatment intended to extend shelf-life. Fruit were immersed in ice-water for 1 h, then treated with or without 100 L L −1 ethylene for 24 h at 24 • C, and finally stored at 20 • C. Fruit firmness, chlorophyll content, ethylene production, respiration rates, contents of pectin, starch and sugar, and the activities of the cell wall modifying enzymes polygalacturonase (PG), pectin methylesterase (PME) and CMCase (cellulase, endo-1,4-␤-glucanase) were analyzed. Total amylase activity was also measured. Immersion in ice-water for 1 h effectively inhibited ripening-associated processes, including peel de-greening and pulp softening during storage or ripening. The delay in ripening was also manifest in reduced ethylene production and respiration rates. The inhibition of softening by cold shock treatment was related to decreased PG and PME activities, that is, retardation of pectin solubilization/degradation. Reduced activities of CMCase and total amylase and conversion of starch to sugar by ice-water immersion also contributed to the delay in softening of harvested banana fruit.

This work aimed at evaluating the effects of sous-vide cooking parameters, such as time and temperature and their interactions, on textural attributes of 'Mondial Gala' and 'Granny Smith' apple cultivars. For this, different response surface methodology-based models were developed. This methodology proved a suitable means for the assessment of changes in textural parameters and cell wall modifications during the processing of apples. 'Mondial Gala' fruit displayed better aptitude for the preservation of textural properties after high-temperature processing conditions and were therefore apparently more suited to sous-vide cooking than 'Granny Smith' apples. Pectin methylesterase activity levels in 'Mondial Gala' apples were enhanced at mild temperatures and pectins in this cultivar displayed a lower degree of methylation. Therefore, the establishment of calcium-mediated linkages between cell wall polymers might have been favoured in 'Mondia...

The behavior of strawberry purees submitted to processes of mild pasteurization (75 °C — 15 s) and severe pasteurization (90 °C — 20 s), using as references fresh and hot-filled purees, was studied. Purees thermally treated were stored at +3 °C during 2 months. Pectin methylesterase activity, rheological behavior, and taste quality were analyzed. The results showed how the pectin methylesterase activity of strawberry puree (0.385nanokatal/mL) was reduced to 25.5% by the mild heat treatment, to 5.4% by the severe one and to undetectable levels in the hot-filled puree. A significant increase in the viscosity was detected as a consequence of the severe thermal treatments, although in any case it decreased with storage time. Taste quality, evaluated by simple ranking tests of samples, was superior in the fresh puree, without significant differences among the three samples thermally treated. The assessors pointed out the greater aromatic intensity of the fresh puree and the lack of cooke...

A hydrophobic coating formulated with maltodextrins, carboxymethylcellulose, propyleneglycol and a mixture of sorbitan esters was applied to pre-climacteric 'Manila' mangoes. The coated fruit were stored for 20 d at 25°C and 80-85% r.h. Changes in mesocarp hardness, as well as pectinesterase (PE), polygalacturonase (PG) and cellulase (Cx) activities were determined. Results during the first 8 d of storage showed evidence that fruit treated with the coating suffered less meso carp softening along with concomitant reductions of PO and Cx activities, than did control fruit. After the initial storage period, activity of PG increased steadily during further ripening of coated fruit.

Ripening of fruit is a very important process but in some fruits early ripening leads to a great damage during long distance transportation. There are various biochemical changes taking place during the phase of ripening of fruit such as changes in respiration, aroma, flavor, ethylene production and activity of cell wall degrading enzymes. Some important cell wall degrading enzymes are Polygalacturonase (PG), Pectin methylesterase (PME), Pectin lyase, RGase. PME is known to act as a cell wall hydrolyzing enzyme, responsible for demethyl esterification of cell wall polygalacturonan. The present study includes the biochemical and molecular characterization of PME from Grand naine variety of Musa acuminata (banana). This study also deals with the in-silico study reflecting inhibition of PME activity in context to delayed ripening in banana. It mainly deals with the identification of a PME1 gene from Grand naine variety of banana. The expression of this gene is related with the process of ripening. The expression of PME1 gene was observed to be peaked on 3 rd day in ethylene treated samples of banana but the activity in untreated samples called control was rather slow and then there was a sudden decrease in their activity in both treated as well as untreated samples. With the help of in-silico study, we observed that banana has maximum homology with carrot by using cross species analysis.The designed model has been reported to be of good quality on the basis of its verification and validation. The designed model was observed to be appropriate for docking. The information of binding sites of ligand provides new insights into the predictable functioning of relevant protein.

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.

Background and Aims In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. † Methods Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. † Key Results A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. † Conclusions By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Pérez-Pérez et al. Cell Wall and Somatic Embryogenesis necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Apple pieces were vacuum-impregnated with either a pectin methylesterase (PME) and calcium solution or with water prior to pasteurization. Pasteurized apple pieces impregnated with PME and calcium showed a significantly higher firmness. Moreover, solid state 13 C NMR spectroscopy of apple cell wall residues revealed an increase of their molecular rigidity. Exogenous PME addition involved a decrease from 82% to 45% of apple pectin degree of methyl-esterification. Microscopic observations of apple slices immunolabelled with antibodies specific for pectins showed that (i) demethyl-esterification was more intense in the cell wall region lining intercellular spaces (demonstrating a key role for these intercellular channels in the enzyme penetration in the tissue during vacuum-infusion) and that (ii) the number of calcium-dimerized deesterified homogalacturonan chains increased. The results corroborate the hypothesis that vacuumimpregnated PME action liberates free carboxyl groups along pectin chains that could interact with calcium, increasing the rigidity of pectins and finally the mechanical rigidity of apple tissue.

Vacuum impregnation is a non-thermal technique used in food technology to replace the air occluded in the products with specific substances, thus modifying food quality. Here, this technique was employed to allow calcium ions to penetrate more efficiently in the tissue structure of diced tomatoes with consequent increase of the product firmness. Calcification was obtained in two ways, that is, either by keeping diced tomatoes at the reduced pressure of 100 mbar or by subjecting the product to cycles of pulsed vacuum from 1013 mbar to 30 mbar. Both procedures were conducted in the presence of calcium chloride solutions at various concentrations and temperatures. The results showed that the highest firmness was obtained by pulsed vacuum calcification at 30 mbar and 45 °C by using 4 cycles of pulsed vacuum. In these conditions, after two-months storage at room temperature, the treated diced tomatoes packaged in tinplate cans showed firmness increase of about 90% with respect to product not treated by vacuum infiltration. These results indicate the pulsed vacuum calcification as a promising process that can be effectively introduced in some phases of the industrial tomato transformation to improve the product texture.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular ...

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation. Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under...

Vacuum impregnation is a non-thermal technique used in food technology to replace the air occluded in the products with specific substances, thus modifying food quality. Here, this technique was employed to allow calcium ions to penetrate more efficiently in the tissue structure of diced tomatoes with consequent increase of the product firmness. Calcification was obtained in two ways, that is, either by keeping diced tomatoes at the reduced pressure of 100 mbar or by subjecting the product to cycles of pulsed vacuum from 1013 mbar to 30 mbar. Both procedures were conducted in the presence of calcium chloride solutions at various concentrations and temperatures. The results showed that the highest firmness was obtained by pulsed vacuum calcification at 30 mbar and 45 °C by using 4 cycles of pulsed vacuum. In these conditions, after two-months storage at room temperature, the treated diced tomatoes packaged in tinplate cans showed firmness increase of about 90% with respect to product not treated by vacuum infiltration. These results indicate the pulsed vacuum calcification as a promising process that can be effectively introduced in some phases of the industrial tomato transformation to improve the product texture.

Textural changes during fruit ripening have been attributed to pectin degradation due to pectic enzymes such as pectinmethylesterase (PME, EC 3.1.1.11). PME catalyzes the de-esterification of pectin, a complex mixture of polysaccharides, namely methyl esterified polygalacturonic acid, with the release of hydrogen and methanol, producing shorter chains causing drastic losses in firmness. Thus, control of PME activity in fruits aiming at texture maintenance and/or improvement is extremely important to the food industry. However, PME activity and properties are dependent on product, environmental and physico-chemical conditions, such as pH and temperature. Therefore, the aim of the present work was to optimize the pectinmethylesterase (PME) enzyme assay from tomato (Solanum lycopersicum 'Zinac') fruits, and determine its kinetics behavior and thermal stability. The highest PME activity was found with a 1.0 M of NaCl extraction solution, with 0.5% citrus pectin, and revealed optima of temperature at 60°C and pH of 8.8. The low k M value (0.011%) for tomato PME describes the high affinity between enzyme and substrate (citrus pectin), whereas the obtained V max value (0.712 U mg-1) relates to the enzyme quantity present in the reaction. The study of PME thermal stability showed two distinct behaviors: an increase of activity from 40 to 50°C and a decrease from 55 to 80°C. At 50°C/10 min an increase in activity up to 17% of activity was observed. At 60°C, about 50% of the activity still remained after heating for 25 min, and PME was completely inactivated at 80°C after 10 min. Data obtained in the temperature range of 55 to 80°C were also satisfactorily described by an Arrhenius first-order kinetic model. These results provide useful information about the different factors that affect tomato PME activity and may be used as a tool for firmness control during postharvest handling and fruit processing.

This study aimed at understanding softening in Jonagold apple (Malus  domestica Borkh.) fruits, by investigating pectin modifications and the evolution of pectin-modifying enzymes during postharvest storage and ripening. Jonagold apples were harvested at commercial maturity and stored at different temperatures and controlled atmosphere conditions for 6 months, followed by exposure to ambient shelf life conditions (20°C under air) for 2 weeks. The composition of the pectic material was analysed. Furthermore, the firmness and the ethylene production of the apples were assessed. Generally, the main changes in pectin composition associated with the loss of firmness during ripening in Jonagold apples were a loss of side chains neutral sugars, increased water solubility and decreased molar mass. Also, the activities of four important enzymes possibly involved in apple softening, b-galactosidase, a-arabinofuranosidase, polygalacturonase and pectin methylesterase, were measured. Pectin-related enzyme activities highly correlated with ethylene production, but not always with pectin modifications.

The behavior of strawberry purees submitted to processes of mild pasteurization (75 °C — 15 s) and severe pasteurization (90 °C — 20 s), using as references fresh and hot-filled purees, was studied. Purees thermally treated were stored at +3 °C during 2 months. Pectin methylesterase activity, rheological behavior, and taste quality were analyzed. The results showed how the pectin methylesterase activity of strawberry puree (0.385nanokatal/mL) was reduced to 25.5% by the mild heat treatment, to 5.4% by the severe one and to undetectable levels in the hot-filled puree. A significant increase in the viscosity was detected as a consequence of the severe thermal treatments, although in any case it decreased with storage time. Taste quality, evaluated by simple ranking tests of samples, was superior in the fresh puree, without significant differences among the three samples thermally treated. The assessors pointed out the greater aromatic intensity of the fresh puree and the lack of cooke...

Apple pieces were vacuum-impregnated with either a pectin methylesterase (PME) and calcium solution or with water prior to pasteurization. Pasteurized apple pieces impregnated with PME and calcium showed a significantly higher firmness. Moreover, solid state 13 C NMR spectroscopy of apple cell wall residues revealed an increase of their molecular rigidity. Exogenous PME addition involved a decrease from 82% to 45% of apple pectin degree of methyl-esterification. Microscopic observations of apple slices immunolabelled with antibodies specific for pectins showed that (i) demethyl-esterification was more intense in the cell wall region lining intercellular spaces (demonstrating a key role for these intercellular channels in the enzyme penetration in the tissue during vacuum-infusion) and that (ii) the number of calcium-dimerized deesterified homogalacturonan chains increased. The results corroborate the hypothesis that vacuumimpregnated PME action liberates free carboxyl groups along pectin chains that could interact with calcium, increasing the rigidity of pectins and finally the mechanical rigidity of apple tissue.

Background and Aims In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. † Methods Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. † Key Results A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. † Conclusions By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/ degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/ degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

We have identi¢ed, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtP-MEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esteri¢cation by inhibition of endogenous PMEs is present in di¡erent plant species.

Background: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. Results: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession "02-5B-318", a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. Conclusions: Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318 R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.

Background: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. Results: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession "02-5B-318", a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318 R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.

Plant infection by a virus is a complex process influenced by virus-encoded factors and host components that 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 cooperate with MP to sustain viral spreading. Activity of PME is modulated by endogenous protein 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 have overexpressed genes encoding two well characterised PMEIs in tobacco and Arabidopsis plants. We here report that in tobacco plants constitutively expressing a PME inhibitor from Actinidia chinensis (AcPMEI) systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms reduced. A delayed movement of Turnip vein clearing virus (TVCV) as well as a reduced susceptibility to the virus was also observed in Arabidopsis plants overexpressing AtPMEI-2. Our results provide evidence that PMEIs are able to limit the tobamovirus movement and to reduce plant susceptibility to the virus.

Background: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. Results: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession "02-5B-318", a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. Conclusions: Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318 R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.