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

Abstract

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <lOVo of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process. INTRODUCTIONPectin methylesterase (PME) is an enzyme found in all plants and in many plant pathogenic bacteria and fungi (RexovaBenkova and Markovic, 1976;Huber, 1983a;Collmer and Keen, 1986). It catalyzes the deesterification of galactosyluronate methyl esters of pectin to their free carboxyl groups. 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). Pectins are primarily composed of polygalacturonic acid (homopolymers of [1-.4]u-~galactopyranosyluronic acid units with varying degrees of the carboxyl groups methylesterified) and rhamnogalacturonan I (a heteropolymer of repeating [1+2]a-L-rhamnosyl-[l~4]a-D-galactosyluronic acid disaccharide units) (Lau et al., 1985). It has been suggested that the carboxyl groups of pectins are highly methylesterified when they are synthesized, but the esters are later cleaved by pectin methylesterase in the cell wall (Kauss and Hassid, 1967;Roberts, 1990). The role of PME in plant cell growth and development is not yet clear. It has been suggested that demethoxylation is necessary for the formation of Ca2+ crossCurrent address: Department of Food Science, Cook College, Rutgers University, New Brunswick, NJ 08903. 2 To whom correspondence should be addressed. bridges in pectins, leading to stabilization of the middle lamella (Fry, 1986;Northcote, 1986). It has been proposed that less methoxylated pectin may be more exten...

A cDNA Clone Highly Expressed in Ripe Banana Fruit Shows Homology to Pectate Lyases

“…Similar results were obtained in tomato fruit in which PME was down-regulated using antisense RNA technology (Tieman et al, 1992;Hall et al, 1993). These results indicate that in addition to PG and PME, other hydrolytic enzymes are likely to be involved in the textural changes associated with ripening.…”

Enhancement of fruit shelf life by suppressingN-glycan processing enzymes

“…Previous studies have shown that polygalacturonase, pectin methylesterase, β-glucanase, and β-galactosidase are not sufficient to significantly impact texture (5)(6)(7)(8)(9)(10)12). This may be due to the presence of functionally redundant components of a complicated metabolic process (6,7,10,13) . It also suggests that the suppression of enzymes acting on cellulose, hemicellulose, and pectin is not sufficient to prevent softening.…”

“…Generally, reduction in fruit firmness due to softening is accompanied by increased expression of cell wall-degrading enzymes acting upon proteins and carbohydrates (4). However, many efforts to suppress expression of cell wall-degrading enzymes have not provided the insight needed to genetically engineer fruits whose softening can be adequately controlled (5)(6)(7)(8)(9)(10)(11). Previous studies have shown that polygalacturonase, pectin methylesterase, β-glucanase, and β-galactosidase are not sufficient to significantly impact texture (5)(6)(7)(8)(9)(10)12).…”

“…However, many efforts to suppress expression of cell wall-degrading enzymes have not provided the insight needed to genetically engineer fruits whose softening can be adequately controlled (5)(6)(7)(8)(9)(10)(11). Previous studies have shown that polygalacturonase, pectin methylesterase, β-glucanase, and β-galactosidase are not sufficient to significantly impact texture (5)(6)(7)(8)(9)(10)12). This may be due to the presence of functionally redundant components of a complicated metabolic process (6,7,10,13) .…”

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit1

“…The model summarizes the data obtained from the above experiments and could be expanded to include other enzymic activities that have been associated with pectin metabolism during ripening, such as pectin methylesterase (Tucker et al, 1982;Harriman et al, 1991;Tieman et al, 1992). A similar model of pectin degradation involving the solubilization of the bulk of pectic polymers independent of both PG and ␤-galactosidase, which act to degrade the solubilized pectins later in softening, has been used to describe cell wall changes in ripening kiwifruit (Redgwell et al, 1992).…”