Polygalacturonase Gene Expression Research Articles

Necrotizing Activity of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum Endopolygalacturonases in Cotton.

Polygalacturonase (PG), which digests the pectin of plant cell walls, contributes to pathogenicity of fungi in plants. To explore the role of PG in pathogenicity of the fungal cotton pathogens Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum, VDPG1 and FOVPG1 were cloned and their expression in different cotton (Gossypium hirsutum) cultivars and media was analyzed. VDPG1 and FOVPG1 were strongly upregulated during infection. Purified VDPG1 and FOVPG1 play important roles in the symptom development of both resistant and susceptible cotton. Moreover, after inoculation with purified PGs, the hydroxyproline content of the cell walls increased in cotton seedlings, with resistant cultivar seedlings showing significantly higher hydroxyproline content than seedlings of the susceptible cultivar. PG gene expression analysis in different media showed that both PG genes were induced in pectin medium but not in glucose medium. This study highlighted the role of VDPG1 and FOVPG1 in pathogenicity and virulence, which were detected in fungus-inoculated cotton, suggesting that PGs play an important role in the pathogenicity of V. dahliae and F. oxysporum f. sp. vasinfectum.

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit.

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit.

Polygalacturonase gene expression and enzymatic activity in salivary glands of laboratory reared and wild populations of Lygus lineolaris (Hemiptera: Miridae)

Lygus lineolaris (Palisot de Beauvois, 1818) (tarnished plant bug) is a serious pest of cotton (Gossypium hirsutum L.) in the Delta region as compared to cotton in the Hills region of the state of Mississippi in USA. The reason for this is unclear but it was hypothesized that the plant cell wall degrading polygalacturonase enzyme system in the salivary glands of L. lineolaris from the Delta could be better adapted for cotton, which is grown more predominantly in the Delta region than in the Hills region. Expression analysis of three primary polygalacturonase genes (LlPG1, LlPG2 and LlPG3) was conducted in laboratory reared and field collected populations of L. lineolaris. Assay of polygalacturonase enzyme activity was also conducted to compare wild collected populations. Initial laboratory and field data revealed gene expression differences in sex, age, region, and host plant which guided the direction of our subsequent study during 2013 and 2014. Based on the results of this study, we propose that the three genes studied may not be reflective of the entire polygalacturonase enzyme system and may not be solely responsible for the observed adaptation of L. lineolaris to cotton in the Delta region than in the Hills region. Analyses also revealed that the expression of the three targeted polygalacturonase genes was affected by the host plant from which the insects were collected and that adults had higher polygalacturonase expression than nymphs. Taken together, our results provide strong evidence for developmental stage specific and host plant based change in expression of PG genes in the salivary glands of L. lineolaris. This, however, was not reflected in total polygalacturonase enzyme activity which was not significantly different between regions, hosts, sex, or developmental stage.

The nascent-polypeptide-associated complex alpha subunit regulates the polygalacturonases expression negatively and influences the pathogenicity of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic plant-pathogenic fungus that infects more than 400 species of plants. In this study the nascent polypeptide-associated complex α subunit gene of S. sclerotiorum (SsNACα; accession No. XP_001593856.1) was cloned and characterized. The relative transcript expression of SsNACα at different morphological stages of asexual development of S. sclerotiorum were analyzed by quantitative real time PCR (qRT-PCR). RNAi-mediated gene silencing was successful for SsNACα, and the mutated strains exhibited less than 15% of the relative expression of SsNACα were obtained and used for studying the biological functions of the gene. A delay in sclerotial maturation for S. sclerotiorum was observed in the SsNACα mutants. The significant elevations for both the activities of pectin-degrading enzymes and the expression of polygalacturonase genes also were associated with the mutated strains, indicating that SsNACα could negatively influence polygalacturonases expression and modulate the pathogenicity of S. sclerotiorum.

Comparative analysis of polygalacturonase in the fruit of strawberry cultivars.

The role of polygalacturonase (PG) in the development, ripening, and softening of fruit from two strawberry cultivars with different flesh firmness and softening characteristics was compared. Changes in PG activity and gene expression during development, ripening and softening were measured. The PG genes from each cultivar were cloned and analyzed, and were classified with other PG genes using phylogenetic analysis. In Toyonoka fruit, PG activity increased gradually, reaching a peak during the pink stage, and remained at this level during post-harvest softening. Changes in PG gene expression were consistent with PG activity in these softer fruits. In the firmer Sweet Charlie fruits, PG activity was detected during the initial development stage, reaching a peak at the white stage, thereafter decreasing gradually with ripening and remaining at this lower level throughout softening. Changes in PG gene expression and PG activity were not consistent in these fruit. For both Toyonoka and Sweet Charlie PG genes (FaTPG and FaSCPG, respectively), the open reading frame was 1218 bp, encoding 405 amino acids. Five different nucleotide sites were observed between the two sequences, leading to two amino acid sequence mutations. FaTPG, FaSCPG, and PG genes from the Fragaria vesca genome were classified into three clades using phylogenetic analysis. The clade containing PG genes involved in fruit softening had functional similarity but there were no functional differences between FaTPG and FaSCPG. Differences in PG activity, gene sequence, and gene expression may have led to different roles of PG during ripening and softening in strawberries with different textures.

Skin pectin metabolism during the postharvest dehydration of berries from three distinct grapevine cultivars

Background and Aims Postharvest dehydration is used to modify certain grape berry quality traits that affect the characteristics of wine. After harvesting, berries undergo several chemical and physical changes that often reflect ongoing or specifically activated metabolic processes, such as the modification of cell wall polymers in the skin. We compared berry skin pectin metabolism during postharvest dehydration in three Italian wine grape cultivars. Methods and Results Corvina, Oseleta and Sangiovese grapes were postharvest dehydrated under the same environmental conditions and displayed different dehydration rates. The quantitative characterisation of pectins by ion-exchange and size-exclusion chromatography revealed that more demethoxylation and depolymerisation occurred in the Corvina berries, which also showed the strongest induction of pectin methylesterase and polygalacturonase gene expression (by microarray analysis) and enzyme activity (by zymography). Conclusions Postharvest dehydration affected the skin pectin metabolism of grape berries to an extent different in each of the three cultivars, reflecting differences in water loss kinetics and metabolic processes. Significance of the Study This is the first description of dynamic cultivar-specific changes in pectin metabolism during the postharvest dehydration of grape berries.

Temporal and spatial expression of polygalacturonase gene family members reveals divergent regulation during fleshy fruit ripening and abscission in the monocot species oil palm

BackgroundCell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process.ResultsThe objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700–5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation.ConclusionsThe expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species.

Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interference

The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity.

Influence of plant growth regulators on polygalacturonase expression in strawberry fruit

Strawberry ( Fragaria × ananassa Duch.) fruit undergoes extensive softening during ripening due to cell wall disassembly, caused by a coordinated action of cell wall proteins and enzymes including polygalacturonases (PGs). The influence of plant growth regulators on PG activity and expression of the corresponding genes has been scarcely analyzed. In this work, the immuno-detection of PG protein (FaPG1) during ripening of strawberry cultivars with contrasting firmness is reported. Two proteins were recognized with molecular mass of 45–50 kDa, which accumulate from 25% red stage in the firmer cultivar (Camarosa) and from white stage in the softer (Toyonoka). The presence of two immunoreactive bands is apparently the product of the differential glycosylation of the same polypeptide. In addition, the effect of different plant growth regulators on the expression of polygalacturonase gene, accumulation of FaPG1, and PG activity was analyzed. The treatment with naphthalene acetic acid (NAA) delayed ripening and anthocyanin accumulation, reduced the expression of FaPG1 and T- PG, the accumulation of FaPG1 protein, and decreased PG activity. The application of ABA increased slightly the expression of PG transcripts, but did not modify significantly the enzyme activity. The treatment with GA 3 reduced anthocyanin accumulation and PG activity, though did not produce remarkable effects on the expression of FaPG1 and T- PG. Anthocyanins increased after treatment with ethephon (ethylene releasing reagent) or sodium nitroprusside (NO providing reagent). The expression of FaPG1 and T- PG increased considerably in response to ethylene and NO. Western-blot data confirmed the trends observed in the expression analysis in response to ethephon and NO, but PG activity was not modified by any of these treatments. However, the application of 1-MCP reduced PG activity, suggesting that ethylene could play a role in the regulation of polygalacturonase expression during strawberry fruit ripening.

Polygalacturonase activity and expression of related genes during ripening of strawberry cultivars with contrasting fruit firmness

Fleshy fruits soften during ripening mainly as a consequence of the catabolism of cell wall components. In strawberry ( Fragaria × ananassa Duch), the depolymerization and solubilization of pectins increase during ripening and contribute to fruit softening. In the present paper, we report the cloning and expression analysis of two polygalacturonase (PG) putative cDNAs: FaPG1 and T-PG. The former seems to be the same sequence of previously reported PG in strawberry, while T-PG cDNA has a deletion of 85 bp that cause a frame shift and would produce an inactive protein. Measurement of total PG activity and expression of FaPG1 and T-PG were performed in strawberry cultivars with contrasting softening rates. The softest cultivar (Toyonaka) showed the higher total PG activity in all ripening stages analyzed. The analysis by RT-PCR revealed that both genes express in the three cultivars, though the expression pattern was different. In the firmer cultivars (Selva and Camarosa) the expression of T-PG was considerably higher than the expression of FaPG1, while the opposite occurred in the softest cultivar (Toyonaka). Therefore, the higher PG activity detected in Toyonaka correlates with the enhanced expression of FaPG1 gene, while the low PG activity found in the firm cultivars correlates with a higher expression of T-PG, a gene that could encode a truncated protein without PG activity. The influence of auxins on both the expression of PG genes and the total PG activity during strawberry fruit ripening was also analyzed.

MONITORING DIFFERENTIAL EXPRESSION DURING FRUIT MATURATION, RIPENING AND STORAGE AS AN IDENTIFICATION TOOL FOR GENE CANDIDATES FOR SUPERIOR APPLE FRUIT QUALITY

A large-scale transcriptional study by means of the cDNA-AFLP technique was performed on skin and flesh tissue of the apple cultivar 'Delbard Jubile' in an attempt to screen for differential gene expression related to fruit maturation and ripening. As such around 600 gene fragments could be visualized of which the expression fluctuated significantly in time. The corresponding profiles were clustered to determine groups of similar expression behavior and related to the expression of ACC-oxidase and polygalacturonase. A selection of profiles was made for isolation and sequencing of the underlying cDNA-fragment. Our results clearly demonstrate that by our approach we visualized genes linked to maturation and ripening as we detected some interesting gene fragment homologies for further characterization. In a second part we tried to describe texture deterioration by following ACC-oxidase and polygalacturonase gene expression during cold storage of 5 cultivars and advanced breeding selections.[...]

657 Genetic Determinants and Control of Fruit Softening

Fruit softening is integral to the ripening process. It is an important component of fruit quality, but also initiates deterioration and is a limiting determinant of shelf-life. Intensive research has attempted to elucidate the biochemical and genetic control of fruit softening with the goal of controlling this process as a means to enhance both fruit quality and shelf-life. Current models of fruit softening focus on cell wall disassembly as the major biochemical event regulating fruit softening. Examination of the sequence of cell wall disassembly in ripening Charentais melon fruit suggested that softening could be divided into two distinct phases. The early stage of fruit softening was associated with the regulated disassembly of xyloglucan polymers and the later softening that accompanies over-ripe deterioration was associated with pectin depolymerization. Characterization of cell wall changes in other fruit, including tomato, suggest that this may represent a general model of sequential cell wall disassembly in ripening fruit. Interestingly, the early events of xyloglucan disassembly were not associated with the activation or expression of xyloclucan hydrolases but were associated with the expression of a ripening-regulated expansin gene. Analysis of transgenic tomato fruit with suppressed expansin gene expression or with suppressed polygalacturonase gene expression supports a general model of sequential disassembly of xyloglucan and pectin that control the early and late phases of fruit softening, respectively.

Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and ethylene production.

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the beta-glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.

Expression of a Water Stress-induced Polygalacturonase Gene in Harvested Cucumber Fruit.

We investigated the molecular response of harvested cucumber (Cucumis sativus L.) fruit to postharvest water stress. Using RT-PCR, we isolated CUPG1 cDNA, a cDNA clone encoding polygalacturonase (PG) that is inducible by water stress in cucumber fruit and characterized the expression of the gene with respect to hormonal control. The full length sequence determined by RACE-PCR, encoded a deduced protein of 435 amino acids that contained several common conserved domains as reported for PGs. The CUPG1 gene expression was also induced by exogenous ethylene but not by the application of abscisic acid (ABA). The level of CUPG1 mRNA induced by water stress, was reduced by 50% in fruits pre-treated with 1-methyl-cyclopropene (MCP), a strong inhibitor of ethylene action, which suggests that the expression of CUPG1 gene could be induced by a direct mechanism through water stress and an indirect mechanism mediated through ethylene produced in response to water stress. We conclude that water loss in harvested cucumber fruit results not only in physical changes but also leads to molecular responses, including PG gene expression.

Utilization of the TEF1-alpha gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae.

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1 alpha, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-alpha protein consisted of 460 amino acids possessing high identify to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A, oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 degrees C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C.

Evaluación de atributos sensoriales y parámetros bioquímicos en frutos de tomate transgénico con reducida actividad de poligalacturonasa / Evaluation of sensory attributes and biochemical parameters in transgenic tomato fruit with reduced polygalacturonase activity

Sensory, physicochemical and biochemical parameters of fresh market tomatoes were monitored in order to determine the nature and extent of quality changes in transgenic tomato fruit with reduced polygalacturonase activity. Acceptability was significantly higher (p < 0.0001) in CR3 transgenic tomatoes than in a commercial long shelf-life hybrid heterozygous for the ripening inhibitor (rin) allele. Preference ratings for CR3 fruits were also significantly superior in some sensory attributes such as colour (p < 0.0002) and flavour (p < 0.0001). A descriptive evaluation of sweetness yielded statistically (p < 0.0143) higher scores for the CR3 control and transgenic fruit, consistent with objec tive measures of soluble solids. Significant increases in survival during longer storage periods were found in polygalacturonase antisense fruits, which were less susceptible to damage or cracking and less prone to infection. In transgenic tomato fruits with reduced polygalacturonase gene expression, the softening rate was not significantly inhibited during ripening but was retarded in the over- ripening stage. Activity assays of α- and β-galactosidases did not show significant differences between antisense and control fruit, except for the β-galactosidase in the breaker stage.

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.

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Temporal Regulation of Polygalacturonase Gene Expression in Fruits of Normal, Mutant, and Heterozygous Tomato Genotypes

Molecular cloning of polygalacturonase (PG; EC 3.2. 1.15) from fruits of tomato (Lycopersicon esculentum Mill cv Rutgers) was accomplished by constructing a cDNA library from turning stage poly(A)(+) RNA in lambdagtll and immunoscreening with polyclonal antibodies raised against purified PG2. Both PG cDNA and antibody probes were used to quantify changes in PG gene expression in pericarp from normal, mutant, and heterozygous genotypes. Results show that PG mRNA, protein, and enzyme activity sequentially peak at the turning, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. PG gene expression was attenuated greatly (0-15% of normal on a gram fresh weight basis) for PG mRNA, protein, and enzyme activity in five ripening-impaired mutants (rin, nor, Nr, Gr, and Long Keeper) tested. Maximum expression of the PG gene in heterozygotes of rin, nor, Nr, Gr, and Long Keeper (crosses with Rutgers) at the mRNA level was about 25, 13, 17, 5, and 62% of the Rutgers turning stage, at the protein level was about 166, 110, 15, 6, and 104% of the Rutgers ripe stage, and at the enzyme activity level was about 69, 37, 4, 1, and 50% of the Rutgers red ripe stage, respectively. No PG gene expression was found in preclimacteric fruits or vegetative tissues. PG mRNA was localized on both free and membrane-bound polyribosomes of ripening pericarp. In addition to transcriptional regulation, mechanisms contributing to mRNA stability, delayed protein accumulation, and posttranslational modifications may play important roles in the overall accumulation of PG activity during fruit ripening.

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.