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

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 <10% 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.

[1]  J. Ricard,et al.  Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 1. A theory of the ionic control of a complex multi-enzyme system. , 1986, European journal of biochemistry.

[2]  P. Hooykaas,et al.  Grown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions. , 1981, Gene.

[3]  D. Hildebrand,et al.  Design and construction of a versatile system for the expression of foreign genes in plants. , 1987, Gene.

[4]  J. Ray,et al.  Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes , 1988, Nature.

[5]  N. Maness,et al.  Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose. , 1990, Analytical biochemistry.

[6]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[7]  Stephen C. Fry,et al.  Cross-Linking of Matrix Polymers in the Growing Cell Walls of Angiosperms , 1986 .

[8]  N. Keen,et al.  The Role of Pectic Enzymes in Plant Pathogenesis , 1986 .

[9]  Liang-Shiou Lin,et al.  Plant cell wall architecture , 1989, Cell.

[10]  G. Tucker,et al.  Differential effects of pectolytic enzymes on tomato polyuronides in vivo and in vitro , 1987 .

[11]  D. Huber.  Polyuronide Degradation and Hemicellulose Modifications in Ripening Tomato Fruit , 1983, Journal of the American Society for Horticultural Science.

[12]  A. Bennett,et al.  ROLE OF CELL WALL HYDROLASES IN FRUIT RIPENING , 1991 .

[13]  A. Kader,et al.  Genotypic Variation for Flavor and Composition in Fresh Market Tomatoes1 , 1977, Journal of the American Society for Horticultural Science.

[14]  J. Mol,et al.  Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. , 1988, BioTechniques.

[15]  W. Hiatt,et al.  Reduction of polygalacturonase activity in tomato fruit by antisense RNA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Northcote.  Control of Pectin Synthesis and Deposition during Plant Cell Wall Growth , 1986 .

[17]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[18]  J. Ray,et al.  Using Antisense RNA to Study Gene Function: Inhibition of Carotenoid Biosynthesis in Transgenic Tomatoes , 1991, Bio/Technology.

[19]  D. Huber,et al.  Comparative Analysis of Pectins from Pericarp and Locular Gel in Developing Tomato Fruit , 1986 .

[20]  J. Mol,et al.  Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. , 1990, The Plant cell.

[21]  J. Labavitch,et al.  A SIMPLIFIED METHOD FOR ACCURATE DETERMINATION OF CELL WALL URONIDE CONTENT , 1978 .

[22]  P. Oeller,et al.  Reversible inhibition of tomato fruit senescence by antisense RNA. , 1991, Science.

[23]  J. Roberts,et al.  Cell Biology of Abscission , 1982 .

[24]  W. Hassid,et al.  Enzymic Introduction of the Methyl Ester Groups of Pectin , 1967 .

[25]  R. Pressey,et al.  Multiple forms of pectinesterase in tomatoes , 1972 .

[26]  M. S. Biggs,et al.  Effect of tunicamycin on in vitro ripening of tomato pericarp tissue , 1985 .

[27]  C. Napoli,et al.  Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. , 1990, The Plant cell.

[28]  P. Albersheim,et al.  Structure of the backbone of rhamnogalacturonan i a pectic polysaccharide in the primary cell walls of plants , 1985 .

[29]  S. Tanksley,et al.  Fine mapping of quantitative trait loci using selected overlapping recombinant chromosomes, in an interspecies cross of tomato. , 1990, Genetics.

[30]  E. Hartree,et al.  Determination of protein: a modification of the Lowry method that gives a linear photometric response. , 1972, Analytical biochemistry.

[31]  D. Tieman,et al.  Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits. , 1991, Plant physiology.

[32]  D. Grierson,et al.  Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants , 1990, Nature.

[33]  N. Carpita,et al.  Measurement of uronic acids without interference from neutral sugars. , 1991, Analytical biochemistry.

[34]  G. Hobson,et al.  The constituents of tomato fruit--the influence of environment, nutrition, and genotype. , 1981, Critical reviews in food science and nutrition.

[35]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[36]  J. Giovannoni,et al.  Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit. , 1990, Plant physiology.

[37]  G. Tucker,et al.  Purification and changes in activities of tomato pectinesterase isoenzymes , 1982 .

[38]  D. Nevins,et al.  Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins. , 1989, Plant physiology.

[39]  M. Scott Biggs,et al.  Biochemical basis of high‐temperature inhibition of ethylene biosynthesis in ripening tomato fruits , 1988 .

[40]  C. Rick,et al.  High soluble-solids content in large-fruited tomato lines derived from a wild green-fruited species , 1974 .

[41]  R. Pressey,et al.  SOLUBILIZATION OF CELL WALLS BY TOMATO POLYGALACTURONASES: EFFECTS OF PECTINESTERASES , 1982 .

[42]  K. Roberts,et al.  Structures at the plant cell surface. , 1990, Current opinion in cell biology.

[43]  M. S. Biggs,et al.  Temporal regulation of polygalacturonase gene expression in fruits of normal, mutant, and heterozygous tomato genotypes. , 1989, Plant physiology.

[44]  H. Lineweaver,et al.  Pectic Enzymes , 1955, Nature.