Molecular Cloning of Tomato Pectin Methylesterase Gene and its Expression in Rutgers, Ripening Inhibitor, Nonripening, and Never Ripe Tomato Fruits

Abstract

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 Xgtil 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 tuming 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.PME3 (EC 3.1.11) is a cell wall-associated protein that demethoxylates pectin to form a carboxylated pectin while releasing methanol and a proton.

An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit.

“…Tomato cotyledons were transformed by cocultivation on tobacco feeder cells as described by Fillatti et al (1987). Transformed plants were selected by growth on kanamycin-containing medium and confirmed by DNAgel blot analysis (Southern, 1975;Sambrooket al, 1989) with 32P-labeled pKYLX7 or a 400-bp EcoRI-Sau3A fragment of the PME cDNA clone (Harriman et al, 1991).…”

“…PME activity has been detected in most plant tissues; however, the enzyme is particularly associated with ripening fruit, abscission zones, and maturing cell walls (Rexova-Benkova and Markovic, 1976;Sexton and Roberts, 1982;Huber, 1983a;Northcote, 1986). lncreases in PME enzyme activity have been reported during ripening of several fruits, including tomato (Tucker et al, 1982;Harriman et al, 1991). Plant cell walls are highly complex structures composed of cellulose, hemicellulose, pectins, structural proteins, and other components (varner and Lin, 1989).…”

“…PME mRNA, protein, and enzyme activity are first detectable in 10-to 20-day-old fruit, and continue to accumulate until the turning stage of fruit ripening before declining (Harriman et al, 1991). At the turning stage of ripening, fruit contain about 20-fold higher levels of PME enzyme activity as compared to 10-day-old fruit (Harriman et al, 1991).To gain insight into the role of PME in tomato fruit development, we introduced an antisense PME gene under the control of the cauliflower mosaic virus 35s promoter into tomato. We…”

“…PME may play a role in fruit softening by increasing in vivo susceptibility of pectins to polygalacturonase during ripening (Pressey and Avants, 1982;Seymour et al, 1987;Koch and Nevins, 1989). Unlike polygalacturonase, which is synthesized only during ripening, PME is produced during the development of tomato fruit (Tucker et ai., 1982;Fischer and Bennett, 1991;Harriman et al, 1991). PME mRNA, protein, and enzyme activity are first detectable in 10-to 20-day-old fruit, and continue to accumulate until the turning stage of fruit ripening before declining (Harriman et al, 1991).…”

Methanol emissions from maize: Ontogenetic dependence to varying light conditions and guttation as an additional factor constraining the flux

“…In leaves, methanol is mainly produced by the demethylation of pectin (Fall and Benson, 1996). Consequently, changes in cell wall structure related to growth (MacDonald and Fall, 1993;Nemecek-Marshall et al, 1995;Galbally and Kirstine, 2002;Karl et al, 2003;Harley et al, 2007), leaf abscission, the ageing of leaf tissues (Harriman et al, 1991) and intercellular air space generation (Nemecek-Marshall et al, 1995) play an important role in methanol emission from leaves. Therefore, methanol fluxes are affected by the seasonality of the vegetation, i.e.…”