Fruit Polygalacturonase Research Articles

Effect of methyl jasmonate on physiological and biochemical quality changes of longkong fruit under low temperature storage

Introduction. Longkong (Aglaia dookkoo Griff.) fruit is a non-climacteric tropical fruit and grows widely in the South-East Asia. It has a unique taste and nutritional properties that make it more valuable to export. However, longkong exhibits a shorter shelf life at ambient (25 °C for 3−5 days) and low temperature (13 °C for 10 days) storage. Therefore, there is an urgent need to extend its shelf life and marketability by using an inexpensive and proficient technique. Materials and methods. Methyl jasmonate (MeJA) treatments with different concentrations (10, 20 and 30 μ Mol L-1 ) were used to control physiological and biochemical quality changes of longkong fruit stored at 13 °C and 85% relative humidity. Fruit with no MeJA treatment served as control. The physiological and biochemical quality analyses were carried out at every four days of the interval. Results and discussion. Longkong pericarp chilling injury (CI) index and ion leakage severely increased in the control fruits as compared with MeJA treated fruit. The increased of pericarp phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) activities were well controlled by MeJA treatments. Fruit polygalacturonase (PG), pectin methyl esterase (PME) and lipoxygenase (LOX) activities were significantly controlled in MeJA treated fruit. Fruit superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities found higher level in the MeJA treated fruit. Conclusion. The different concentrations of MeJA treatment effectively reduced the severity of physiological and biochemical quality changes in longkong fruit under prolonged low temperature storage.

Combined Effects of Pre-cooling, Application of Natural Extracts and Packaging on the Storage Quality of Apple (<i>Malus domestica</i>) cv. Royal Delicious

During recent years, global concern for protection of the environment has led researchers to investigate the use of natural flora as one of the sources for crop protection. The present investigation was also directed to find out the natural and ecofriendly fruit coating, package and storage alternatives for the extension of the storage life of apple (<em>Malus domestica</em>) cv. 'Royal Delicious'. Surface coating of apple with 1, 1.5, 2% neem oil (<em>Azadirachta indica</em>) and 10, 15, 20% extracts of marigold flower (<em>Tagetes erectus</em>), along with pre-cooling were tested on the storage quality of the fruit. The fruits were analyzed for physicochemical and physiological characters such as loss in weight, fruit firmness, total soluble solids (TSS) content, titratable acidity (TA), pH, content of reducing sugars, pectin, total anthocyanin and polygalacturonase (PG) activity. They were also analyzed for fruit spoilage. The results revealed that, surface-coating of apple with 1.5- 2% concentration of neem oil along with pre-cooling was the most effective in providing better physico-chemical umbrella, effectively lowering fruit softening and PG enzyme activity, and significantly lowering fruit spoilage during storage. Similarly, packaging of fruits with corrugated fiber board (CFB) boxes + paper mould trays, CFB + Polyethylene (PE) liners and shrink-wrapped tray packing during storage (2±1°C and 85-90% RH), showed that the surface-coating of apple with 2% neem oil together with shrink-wrapped tray packing provided better retention of storage life of fruit and the treatment effect on physico-chemical characteristics of fruits were significant. <br /> DOI: <a href="http://dx.doi.org/10.4038/tar.v21i1.2582">http://dx.doi.org/10.4038/tar.v21i1.2582</a> <em>Tropical Agricultural Research</em> Vol. 21(1): 10-20 (2009)

P.2.d.003 The omega-3 polyunsaturated fatty acid docosahexaenoic acid suppresses neuroinflammatory responses and induces heme oxygenase-1 expression in BV-2 microglia

Persimmon (Diospyros kaki cv. Jiro) was transformed with the gene encoding the pear fruit (Pyrus communis) polygalacturonase inhibiting protein (PGIP) using Agrobacterium tumefaciens EHA101. Two plasmid constructions were used for transformation; pDU94.0928 containing chimeric genes of PGIP, GUS and NPTII in its T-DNA region, and pYS95.091 containing only the GUS and NPTII sequences (control transformations). Among 165 callus lines obtained from infection by A. tumefaciens with pDU94.0928, 15 showed GUS activity. A PGIP gene fragment of 132 bp was amplified by PCR in nine of 15 callus lines. Shoots were regenerated from three of the nine PGIP-containing callus lines. The transformation of the regenerated shoots was confirmed by PCR and Southern blot analyses using NTPII, GUS and pear PGIP probes. PGIP expression in shoot tissues was analyzed by western blot analysis. The protein expression was observed in all three shoot lines regenerated from transformed calli with the PGIP gene. Fungal PG activity was inhibited by crude extracts from shoots transformed with the PGIP gene substantially more than by those from the control transformants obtained from the transformation with pYS95.091.

Cell wall disassembly during papaya softening: Role of ethylene in changes in composition, pectin-derived oligomers (PDOs) production and wall hydrolases

Cell wall disassembly in ripening climacteric fruit is a highly complex process where ethylene plays a crucial role. Ethylene inhibitors can be used to explore the changes in the cell wall matrix and cross-linked polysaccharides in ethylene-regulated processes. The results of applying the ethylene receptor blocking inhibitor 1-methylcyclopropene (1-MCP) and the ethylene-releasing compound ethephon (2-chloroethylphosphonic acid) indicate that softening of ‘Maradol’ papaya fruit is dependent on ethylene. When fruit were induced to ripen extensively by exposure to a high dose of ethephon, 1-MCP inhibited the subsequent softening dramatically, but when inhibition of the ethylene response was caused by application of 1-MCP, subsequent fruit treatment with ethephon promoted extensive loss of galactose from the water-soluble polysaccharides, but this was not accompanied by fruit softening. The cell wall changes accompanying normal fruit softening were pectin solubilization and polyuronide depolymerization and these processes occurred simultaneously. Polygalacturonase likely is responsible for the ripening-associated changes in ‘Maradol’ papaya fruit texture and pectin polymer integrity. An increase in extractable fruit polygalacturonase follows the increased presence of pectin-derived oligosaccharides.

Influence of aqueous 1-methylcyclopropene concentration, immersion duration, and solution longevity on the postharvest ripening of breaker-turning tomato ( Solanum lycopersicum L.) fruit

The influence of aqueous 1-methylcyclopropene (1-MCP) concentration, immersion duration, and solution longevity on the ripening of early ripening-stage tomato ( Solanum lycopersicum L.) has been investigated. Tomato fruit at the breaker-turning stage were fully immersed in aqueous 1-MCP at 50, 200, 400 and 600 μg L −1 for 1 min, quickly dried, and then stored at 20 °C. Ethylene production, respiration, surface color development, and rate of accumulation of lycopene and polygalacturonase (PG) activity were suppressed and/or delayed in fruit exposed to aqueous 1-MCP. Suppression of ripening was concentration dependent, with maximum inhibition in response to 1 min immersion occurring at concentrations of 400 and 600 μg L −1. Climacteric ethylene peaks were delayed approximately 6, 7, and 9 d and respiration was strongly suppressed in fruit treated with aqueous 1-MCP at 200, 400, and 600 μg L −1, respectively, compared with control fruit. Fruit firmness, lycopene content, PG activity, and surface hue of fruit treated at the three higher levels remained strongly suppressed compared with control. Skin hue values and pericarp lycopene content in response to treatment at the subthreshold 50 μg L −1 provided evidence for differential ripening suppression in external versus internal tissues. Maximum delay of softening and surface color development in response to 50 μg L −1 aqueous 1-MCP occurred following immersion periods of between 6 and 12 min. Factors affecting fruit penetration by aqueous 1-MCP and mechanisms contributing to recovery from 1-MCP-induced ripening inhibition are discussed.

Transformation of persimmon with a pear fruit polygalacturonase inhibiting protein (PGIP) gene

Persimmon ( Diospyros kaki cv. Jiro) was transformed with the gene encoding the pear fruit ( Pyrus communis) polygalacturonase inhibiting protein (PGIP) using Agrobacterium tumefaciens EHA101. Two plasmid constructions were used for transformation; pDU94.0928 containing chimeric genes of PGIP, GUS and NPTII in its T-DNA region, and pYS95.091 containing only the GUS and NPTII sequences (control transformations). Among 165 callus lines obtained from infection by A. tumefaciens with pDU94.0928, 15 showed GUS activity. A PGIP gene fragment of 132 bp was amplified by PCR in nine of 15 callus lines. Shoots were regenerated from three of the nine PGIP-containing callus lines. The transformation of the regenerated shoots was confirmed by PCR and Southern blot analyses using NTPII, GUS and pear PGIP probes. PGIP expression in shoot tissues was analyzed by western blot analysis. The protein expression was observed in all three shoot lines regenerated from transformed calli with the PGIP gene. Fungal PG activity was inhibited by crude extracts from shoots transformed with the PGIP gene substantially more than by those from the control transformants obtained from the transformation with pYS95.091.

Transgenic expression of pear PGIP in tomato limits fungal colonization.

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

Molecular characterization of a tomato polygalacturonase gene abundantly expressed in the upper third of pistils from opened and unopened flowers.

A polygalacturonase (PG) gene, TPG7 (Lyces;Pga1;8), has been cloned from tomato (Lycopersicon esculentum Mill., cv. Rutgers). RNA blot analysis reveals that TPG7 is highly expressed in pistils (ovary removed) from unopened and fully open flowers. Dissection of mature pistils demonstrated that TPG7 expression is limited to the top third (stigmatic region) of the pistils. This is contrasted with another tomato PG, TAPG4, which is also expressed in the same region of the pistil but only in mature pistils from fully open flowers. Hybridization of the TPG7 probe to anther RNA was nil to none and was barely detectable in RNA from leaf and flower abscission zones. The TPG7 polypeptide shares 39% sequence identity with the tomato fruit PG and between 63% and 73% sequence identities with six other tomato PGs.

Fruit-specific lipoxygenase suppression in antisense-transgenic tomatoes

Abstract To determine the importance of the enzyme lipoxygenase (LOX) in the generation of volatile C6 aldehyde and alcohol flavour compounds, two antisense LOX genes were constructed and transferred to tomato plants. The first of these constructs (p2ALX) incorporated the fruit-specific 2A11 promoter [Van Haaren, M.J.J., Houck, C.M., 1993. A functional map of the fruit-specific promoter of the tomato 2A11 gene. Plant Mol. Biol. 21, 625–640] and a 1.2 Kb antisense fragment of the cDNA of PTL1 (TomloxA) [Ferrie, B.J., Beaudoin, N., Burkhart, W., Bowsher, C.G., Rothstein, S.J., 1994. The cloning of two tomato lipoxygenase genes and their differential expression during tomato fruit ripening. Plant Physiol. 106, 109–118]; no terminator was included. The second construct (pPGLX) consisted of the ripening-specific polygalacturonase (PG) promoter and terminator [Nicholass, F.J., Smith, C.J.S., Schuch, W., Bird, C.R., Grierson, D., 1995. High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol. Biol. 28, 423–435] and a 400 bp antisense fragment of the tomato cDNA of PTL1 (TomloxA). Both constructs included the highly conserved LOX region (approximately 110 bp) shared by all plant and mammalian lipoxygenase genes. Reduced levels of endogenous TomloxA and TomloxB mRNA (2–20% of wild-type) were detected in transgenic fruit containing the p2ALX construct compared to non-transformed plants, whereas the levels of mRNA for a distinct isoform, TomloxC, were either unaffected or even increased. The pPGLX construct was much less effective in reducing endogenous LOX mRNA levels. In the case of the p2ALX plants, LOX enzyme activity was also greatly reduced compared with wild-type plants. Analysis of flavour volatiles, however, indicated that there were no significant changes. These findings suggest that either very low levels of LOX are sufficient for the generation of C6 aldehydes and alcohols, or that a specific isoform such as TomloxC, in the absence of TomloxA and TomloxB, is responsible for the production of these compounds.

Differential expression of a polygalacturonase gene family in Arabidopsis thaliana.

By systematic sequencing of a flower bud cDNA library from Arabidopsis thaliana, we have identified four cDNAs encoding polygalacturonase. The corresponding genes, together with seven other A. thaliana genes present in the databases, form a small gene family. Sequence comparisons of the deduced polypeptides within the gene family or with other plant polygalacturonases allow classification of the genes into different clades. Five polygalacturonases, including all those isolated from the flower buds, are closely related to the enzyme in pollen. Of the six remaining polygalacturonases, three are more closely related to the abscission-specific type of enzyme and two others to the fruit polygalacturonase. The last one is more distantly related to the others and might correspond to a new type of polygalacturonase. Expression of the different genes was analysed on Northern blots and by a PCR-based strategy. Results indicate that if, as expected, the cDNAs isolated from the flower bud library are strongly expressed in pollen, other genes are expressed at a low level in young developing tissues, such as in seedlings and roots, suggesting that they could be implicated in the cell wall modifications observed during cell elongation and/or expansion which occur in these tissues.

Genomic organization of six tomato polygalacturonases and 5' upstream sequence identity with tap1 and win2 genes.

Recently, three polygalacturonase (PG) cDNAs (TAPG1, TAPG2, and TAPG4) were identified in a library prepared from tomato (Lycopersicon esculentum cv. Rutgers) leaf abscission zones. Genomic clones encoding these three cDNAs have been identified. Moreover, the genomic clones include three additional PG genes, TPG3, TAPG5 and TPG6, which have not been previously reported. A transcript for TAPG5 was detected in the RNA from leaf and flower abscission zones; however, transcripts for TPG3 and TPG6 were not. DNA sequence analysis revealed that TAPG1, TAPG2, and TPG3 are linked in a close tandem array. TAPG4, TAPG5 and TPG6 are also closely linked to each other but in divergent and inverted orientations and are not closely linked to TAPG1, TAPG2, or TPG3. TAPG4, TAPG5 and TPG6 map to the middle of chromosome 12. TPG6 contains two introns. The other five PG genes include four exons and three introns. The relative positions of introns 1 and 2 are shared by all six PG genes. The position of intron 3 is conserved in the other five. The structure of the tomato fruit PG gene, which contains 8 introns, is compared with that of the six PG genes described above. Of interest is an approximately 300 bp inverted repeat found in TAPG1, TAPG2 and TAPG4 that shares significant sequence identity with sequence in the first intron of the tomato anionic peroxidase gene, tap1. RNA blot analysis indicates that the transcript for an anionic peroxidase increases during abscission. In addition, a 250 bp sequence found in TPG3 shares high sequence identity with a 5' upstream region in a wound-induced win2 gene from potato. Potential sites of transcriptional regulation in these genes are discussed.

Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: A Re-examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 L/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 L/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 L/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 L/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 L/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.

High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions.

The 1.4 kb 5' polygalacturonase (PG) gene-flanking region has previously been demonstrated to direct ripening-specific chloramphenicol acetyl transferase (CAT) expression in transgenic tomato plants. The steady state level of CAT mRNA in these plants was estimated to be less than 1% of the endogenous PG mRNA. Further constructs containing larger PG gene-flanking regions were generated and tested for their ability to direct higher levels of reporter gene expression. A 4.8 kb 5'-flanking region greatly increased levels of ripening-specific reporter gene activity, while a 1.8 kb 3' region was only shown to have a positive regulatory role in the presence of the extended 5' region. Transgenic plants containing the CAT gene flanked by both of these regions showed the same temporal pattern of accumulation of CAT and PG mRNA, and steady-state levels of the transgene mRNA were equivalent to 60% of the endogenous PG mRNA on a per gene basis. The proximal 150 bp of the PG promoter gave no detectable CAT activity. However, the distal 3.4 kb of the 4.8 kb 5' PG promoter was shown to confer high levels of ripening-specific gene expression when placed in either orientation upstream of the 150 bp minimal promoter. The DNA sequence of the 3.4 kb region revealed a 400 bp imperfect reverse repeat, and sequences which showed similarity to functionally significant sequences from the ripening-related, ethylene-regulated tomato E8 and E4 gene promoters. The possible roles of the flanking regions in regulating PG gene expression are discussed.

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.

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit.

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.

Simultaneous Inhibition of Two Tomato Fruit Cell Wall Hydrolases, Pectinmethylesterase and Polygalacturonase, with Antisense Gene Constructs

The cloning and sequencing of two cDNAs representing pectinmethylesterase (PME) RNAs from tomato fruit is reported. The clones were used to construct chimeric antisense PME genes designed for high-level constitutive expression in plants. A full-length antisense PME gene construct, in conjunction with a chimeric antisense polygalacturonase gene, was introduced into tomato plants via Agrobacterium-mediated transformation. Simultaneous and significant reduction in the mRNA and protein levels of these normally highly abundant cell wall hydrolases of the pectin degradation pathway were observed in ripe fruit of transformants. Thus, antisense gene constructs in plants can be used to block multiple steps in metabolic pathways simultaneously.

The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.

The b Subunit of Tomato Fruit Polygalacturonase Isoenzyme 1: Isolation, Characterization, and Identification of Unique Structural Features

The b Subunit of Tomato Fruit Polygalacturonase Isoenzyme 1: Isolation, Characterization, and Identification of Unique Structural Features

MOLECULAR CHARACTERIZATION OF A POLYGALACTURONASE INHIBITOR OF PEAR FRUIT

Higher plant inhibitors of fungal polygalacturonases are potential contributors to plant defense. To test this hypothesis we have raised antibodies against the `Bartlett' pear fruit polygalacturonase inhibitor (PGIP) and cloned a pear fruit PGIP cDNA. The pear PGIP cDNA was isolated by polymerase chain reactions based on our amino acid and nucleotide sequence information. Sequence analysis predicts a gene product of 34.5 kD with an isoelectric point of 6.02 in agreement with our biochemical data. Seven potential glycosylation sites are consistent with the glycoprotein character of these PGIPs. Southern blot analysis suggests the presence of 1 or 2 genes in the pear genome. Northern blot analysis indicates the presence of a transcript of 1.5 kb. Western blot analysis shows cross-reactivity of the anti-pear PGIP antibody to various dicot species as well as corn.