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.     

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.     

Effect of Sodium Chloride on Fruit Ripening of the Nonripening Tomato Mutants nor and rin

Tomato (Lycopersicon esculentum Mill) plants of the nonripening mutant nor, the ripening-inhibited mutant rin, and the normal cultivar `Rutgers' were grown in nutrient solution supplemented with 3 grams per liter NaCl from the time of anthesis. In plants treated with NaCl, all the ripening parameters of the fruits of the nor mutant increased, but those of the rin mutant did not. The ripening of the fruits of the NaCl-treated nor plants was characterized by the development of a red color and taste, increased pectolytic activity, and increased evolution of CO₂ and ethylene. These changes do not normally take place in nor under control conditions. The values of these ripening parameters in nor were lower than those of the normal Rutgers fruits. In addition, both in nor and rin and in the normal variety, exposure of the plants to NaCl shortened the developmental period of the fruit, decreased the fruit size, and increased the concentrations of total soluble solids, Na⁺, Cl⁻, reducing sugars, and titratable acids in the fruit. The role of NaCl in overcoming the inability of nor to ripen is discussed.     

Resistance of rin and nor tomato mutants to postharvest Rhizopus infection

Fruits of the two non-ripening mutants of tomato, rin and especially nor, were markedly more resistant to Rhizopus stolonifer infection than the normal Rutgers fruit. Following artificial inoculations by contact with a diseased normal tomato covered with mycelium and sporangia, no infection of unwounded nor fruit occurred at its mature-green stage. At the mature stage the resistance of nor mutant fruit was manifested by a prolongation of the incubation period of the disease as well as by a markedly reduced incidence of rotted fruits. Chilling injury of fruit, prior to spore inoculation, was found to be a good means for indicating the relative resistance of the mutants as compared with the normal tomato. The relationship between the resistance of the mutant tomatoes to Rhizopus infection and their response to induced peel damage as a result of the contact or the chilling procedure, led to the assumption that fruit resistance is associated with the inability of the fungus to penetrate the periderm, rather than with fungal development within the fruit.     

Stock-Scion Interactions of Normal and Fruit Ripening Mutants rin and nor in Tomato

Scions of the non-ripening rin and nor tomato strains (Lycopersicum esculentum Mill.) were grafted on normal understock plants (cv. Rutgers) in an effort to study the influence of roots and vegetative tissue on the ripening behavior of the tomato fruit. Receiprocal grafts of ‘Rutgers’ scions on rin and nor understocks as well as grafted and ungrafted controls were also established. No alteration in the ethylene, and CO₂ evolution and color development of either mutant fruits on normal understock or of normal fruits on mutant understock occurred. We suggest that the inability of rin and nor mutant fruits to ripen normally stems either from the presence in mutant fruit of a non-translocatable ripening inhibitor, or from the absence of a non-translocatable ripening factor.     

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.     

Composition of ethanol-insoluble polysaccharides in water extracts of ripening tomatoes

The carbohydrate composition of the 80% ethanol-insoluble polysaccharides (EIP) from water extracts of ‘Rutgers,’ rin (ripening inhibitor) and nor (non-ripening) tomatoes has been determined. The amount of EIP extracted from ‘Rutgers’ fruit increased from 0.34 to 0.61 mg/g fr. wt during ripening little change occurred in rin or nor fruit. The carbohydrate composition (μg/g fr. wt) of EIP from mature green fruit was: galacturonic acid (48); rhamnose (3); arabinose (20); xylose (48); mannose (31); glucose (139); galactose (51). The most obvious changes that accompanied ripening were a 7.4-fold and 4-fold increase in galacturonic acid and rhamnose content, respectively. These changes were attenuated in the ripening mutants. EIP was fractionated into three major peaks by using DEAE-cellulose ion exchange chromatography. The first peak, which was not retained by the column, contained predominantly glucose and mannose, with lower amounts of galacturonic acid and galactose. The two retained peaks which eluted at 0.1 and 0.2 M sodium chloride contained primarily galacturonic acid, xylose, galactose and arabinose. The galacturonic acid content of these two fractions increased substantially during ripening, whereas the other components decreased. No changes were evident in the ripening mutants. No increase in water-soluble polysaccharides high in galactose content was observed during ripening.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

Polygalacturonase and Cellulase Enzymes in the Normal Rutgers and Mutant rin Tomato Fruits and Their Relationship to the Respiratory Climacteric

Cell wall enzymes at different stages of fruit development were compared between the normal Rutgers and the isogenic nonripening rin tomato. In Rutgers, a detectable increase in polygalacturonase (PG) activity was observed 6 days prior to the respiratory climacteric (43 days postanthesis). The maximum increase in PG activity occurred after C₂H₂ and CO₂ production reached their peak. However, in the rin tomato, no change in PG activity was noted up to 100 days postanthesis. Cellulase activity increased in Rutgers fruits prior to the respiratory climacteric and continued to increase thereafter. Similar changes in cellulase activity were also observed in the nonclimacteric rin fruits. Short term ethylene treatment (2 days) of 36-day-old rin fruits increased cellulase activity, but had no effect on PG activity. Detectable changes in other parameters of ripening, such as chlorophyll loss and softening, also occurred prior to the respiratory climacteric. These results suggest that the failure of rin fruits to ripen is related to their low PG activity during maturity as compared with normal fruits.     

Pectolytic and Cellulolytic Activity of Botrytis cinerea Pers. Related to Infection of Non-ripening Tomato Mutants

Enzymes of Botrytis cinerea were detected in vitro using various carbon sources. Pectin-pectate as a sole carbon source induced both polygalacturonase (PG) and pectin lyase (PL) activity, whereas carboxymethylcellulose served as an inducer for cellulase (C_x) activity. PG activity appeared earlier than C_x activity when induced by their respective sources. Both PG and PL activities were detected earlier and their level was higher on cell walls of the normal tomato fruit, than of the nor mutant, and in each case activity was higher on cell walls of the mature fruits than of the mature-green ones. Whereas relatively high rates of PG and PL activity were recorded on autoclaved tomato homogenate (TH) of both the normal and the nor fruits, only trace levels of PG activity were recorded on unautoclaved media, except for those prepared from ripe normal fruits, and no PL activity was detected on either of the unsterilized media. Botrytis-infection resulted in PG activity in the enzyme-less rin and nor mutant fruits at both stages of maturity and in the normal and hybrid fruits at their mature-green stage. In the ripe normal and hybrid fruits, infection increased the level of PG activity recorded prior to inoculation. An association was drawn between the low PG activity recorded in the nor mutant and its hybrid at initial stages of invasion and their resistance to infection. Following infection an increase in the level of C_x activity over that recorded in healthy fruits was found in all the tomato genotypes, whereas no PL was recorded in either healthy or infected fruits.     

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.     

Initiation at the flowering stage of postharvest Botrytis stem-end rot in normal and non-ripening tomato fruits

Flowers of a normal tomato cultivar and of the two non-ripening mutants rin and nor, were sprayed with a Botrytis cinerea spore suspension. Stem-end infection developed in 84–100% of the harvested fruits of these sprayed flowers when held in high r.h. In both nor and rin fruits, rot development remained restricted to the stem-end area and fruit shoulders, whereas in the normal fruit decay spread rapidly from the stem-end over the entire fruit. Spraying the flowers with iprodione prior to spore inoculation resulted in decreased incidence of decay and suppressed hyphal growth and sporulation in all types of fruits. The results indicate that floral organs serve as a pathway for B. cinerea stem-end initiation in both the normal and the mutant fruit and suggest that this mode of penetration is not related to the marked resistance to infection attributed to the mutant fruits.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.     

Association between Elemental Content and Fruit Ripening in rin and Normal Tomatoes

Analysis of Ca and other inorganic ions in the pericarp of rin, a nonripening mutant, and normal tomato (Lycopersicon esculentum Mill) fruits revealed significant differences in their accumulations at advanced stages of fruit development. During early stages of fruit development, soluble Ca was higher in Rutgers and there were no detectable changes in the accumulation patterns of the other inorganic ions. In the mutant rin, bound Ca continued to increase with age and it was twice as high as compared to earlier stages. In the normal tomato, bound Ca decreased about 3-fold at later stages of development. Mg and Mn also showed some changes similar to Ca. K continued to increase with age and the mutant rin had lower levels than Rutgers throughout development. Other ions such as P, Zn, Cu, and Co were similar in the mutant and normal fruits. These results are interpreted as indicating that high levels of bound divalent cations in the mutant rin may be associated with an altered membrane and cell wall and play a role in fruit ripening.     

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.