Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant.     

Differential Expression of Two Endo-1,4-[beta]-Glucanase Genes in Pericarp and Locules of Wild-Type and Mutant Tomato Fruit

The mRNA accumulation of two endo-1,4-[beta]-D-glucanase genes, Cel1 and Cel2, was examined in the pericarp and locules throughout the development of normal tomato (Lycopersicon esculentum) fruit and the ripening-impaired mutants rin and Nr. Both Cel1 and Cel2 were expressed transiently at the earliest stages of fruit development during a period corresponding to cell division and early cell expansion. In the pericarp, the mRNA abundance of both genes increased markedly at the breaker stage; the level of Cel1 mRNA decreased later in ripening, and that of Cel2 increased progressively. Cel2 mRNA levels also increased at the breaker stage in locules but after initial locule liquefaction was already complete. In rin fruit mRNA abundance of Cel1 was reduced and Cel2 was virtually absent, whereas in Nr Cel1 was expressed at wild-type levels and Cel2 was reduced. In wild-type fruit ethylene treatment slightly promoted the mRNA accumulation of both genes. In rin fruit ethylene treatment strongly increased the mRNA abundance of Cel1 to an extent greater than in wild-type fruit, but Cel2 mRNA was absent even after ethylene treatment. These two endo-1,4-[beta]-D-glucanase genes, therefore, do not show coordinated expression during fruit development and are subject to distinct regulatory control. These results suggest that the product of the Cel2 gene contributes to ripening-associated cell-wall changes.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.     

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a λgtll expression library constructed from MG pericarp poly(A)⁺ RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.     

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.     

Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a Role for Polygalacturonase in Ripening-Associated Pectin Disassembly

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.Fruit ripening is a genetically programmed event that is characterized by a number of biochemical and physiological processes that alter fruit color, flavor, aroma, and texture (Brady, 1987). Extensive cell wall modifications occur during ripening and are thought to underlie processes such as fruit softening, tissue deterioration, and pathogen susceptibility. These modifications are regulated at least in part by the expression of genes that encode cell wall-modifying enzymes (Fischer and Bennett, 1991). Pectins are a major class of cell wall polysaccharides that are degraded during ripening, undergoing both solubilization and depolymerization. In tomato the majority of ripening-associated pectin degradation is attributable to the cell wall hydrolase PG. Transgenic tomato plants with altered PG gene expression indicated that PG-dependent pectin degradation is neither required nor sufficient for tomato fruit softening to occur (Sheehy et al., 1988; Smith et al., 1988; Giovannoni et al., 1989). However, data from experiments using fruit of the same transgenic lines strongly suggested that PG-mediated pectin degradation is important in the later, deteriorative stages of ripening and in pathogen susceptibility of tomato fruit (Schuch et al., 1991; Kramer et al., 1992).In melon (Cucumis melo) substantial amounts of pectin depolymerization and solubilization take place during ripening (McCollum et al., 1989; Ranwala et al., 1992; Rose et al., 1998), implicating a role for PG in ripening-associated cell wall disassembly in melons. However, melons have been reported to lack PG enzyme activity (Hobson, 1962; Lester and Dunlap, 1985; McCollum et al., 1989; Ranwala et al., 1992). The possibility exists that PG is present in melon but that it does not conform to the expected enzymic properties in terms of abundance and/or lability, a point illustrated by recent reports in apple and strawberry, which were previously reported to lack PG activity but that do in fact accumulate low amounts of protein and/or measurable activity (Nogata et al., 1993; Wu et al., 1993). In light of the unexplained discrepancy between ripening-associated pectin depolymerization and undetectable PG activity in melons, we have undertaken a study to reexamine the status of PG in melon using the rapidly ripening cv Charentais (C. melo cv Reticulatus F1 Alpha).As reported for other cultivars, Charentais melons exhibit substantial solubilization and a downshift in the molecular-mass profile of water-soluble pectins, but this is associated with the later stages of ripening, after softening is initiated (Rose et al., 1998). By utilizing a molecular approach to analyze PG in melon, we have attempted to overcome some of the potential limitations of biochemical methods, such as low abundance of protein, reliance on other cell wall components, and unknown cofactors for activity and/or lability during extraction. In doing so, we have identified and characterized a multigene family encoding putative PGs from Charentais melon, including three PG homologs that are expressed abundantly during fruit ripening. The pattern of PG gene expression correlates temporally with the depolymerization of water-soluble pectins and an increase in pectin-degrading enzyme activity. Three additional PG homologs were also identified and shown to be expressed in mature anthers and fruit-abscission zones, tissues that, similar to ripening fruit, are undergoing cell separation. The most abundant ripening-associated putative PG mRNA, MPG1, was expressed in the filamentous fungus Aspergillus oryzae. The culture filtrate from the transformed A. oryzae strain XMPG1 exhibited endo-PG activity, further supporting a role for endo-PG in ripening-associated pectin disassembly in Charentais melon fruit.     

Changes in Gene Expression during Tomato Fruit Ripening

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)⁺ and poly(A)⁻ RNAs. Peak levels of total RNA, poly(A)⁺ RNA, and poly(A)⁺ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)⁺ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)⁺ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.     

Transplantation Studies with Immature Fruit of Normal, and rin and nor Mutant Tomatoes

The aim of the work reported herein was to determine whether the lack of normal ripening in fruits of rin and nor tomato mutants is due to the presence of ripening inhibitors or to the lack of ripening factors in the fruit. A fruit tissue transplantation technique was developed for this purpose.     

Organization and expression of polygalacturonase and other ripening related genes in Ailsa Craig “Neverripe” and “Ripening inhibitor” tomato mutants

The organization and expression of ripening-related genes were investigated in normal tomato (Lycopersicon esculentum cv. Ailsa Craig) and in Neverripe (Nr) and Ripening inhibitor (rin) mutants.Hybridization studies with ripening-related cDNA clones showed that the gene for polygalacturonase (PG) is barely expressed in rin and expressed at a low level in Nr fruit. Four other genes were found to be expressed at reduced levels in rin. Exogenous ethylene was able to restore higher levels of expression of all the genes showing reduced expression in rin except that for PG. However, exogenous ethylene did not restore normal ripening in rin fruit. Analysis of chromosomal DNA by Southern blotting indicated that all the genes studied, including the PG gene, and also an upstream promoter of the PG gene, are present in the rin and Nr genomes and appear to be arranged in a similar way to those in normal tomatoes. The results are discussed in the light of the suggestion that these mutations may involve part of the regulatory apparatus leading to the expression of ripening genes such as PG.     

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.     

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 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/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 μL/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 μL/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 μL/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.