Abstract
Molecular cloning of polygalacturonase (PG; EC 3.2. 1.15) from fruits of tomato (Lycopersicon esculentum Mill cv Rutgers) was accomplished by constructing a cDNA library from turning stage poly(A)+ RNA in λgtll and immunoscreening with polyclonal antibodies raised against purified PG2. Both PG cDNA and antibody probes were used to quantify changes in PG gene expression in pericarp from normal, mutant, and heterozygous genotypes. Results show that PG mRNA, protein, and enzyme activity sequentially peak at the turning, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. PG gene expression was attenuated greatly (0-15% of normal on a gram fresh weight basis) for PG mRNA, protein, and enzyme activity in five ripening-impaired mutants (rin, nor, Nr, Gr, and Long Keeper) tested. Maximum expression of the PG gene in heterozygotes of rin, nor, Nr, Gr, and Long Keeper (crosses with Rutgers) at the mRNA level was about 25, 13, 17, 5, and 62% of the Rutgers turning stage, at the protein level was about 166, 110, 15, 6, and 104% of the Rutgers ripe stage, and at the enzyme activity level was about 69, 37, 4, 1, and 50% of the Rutgers red ripe stage, respectively. No PG gene expression was found in preclimacteric fruits or vegetative tissues. PG mRNA was localized on both free and membrane-bound polyribosomes of ripening pericarp. In addition to transcriptional regulation, mechanisms contributing to mRNA stability, delayed protein accumulation, and posttranslational modifications may play important roles in the overall accumulation of PG activity during fruit ripening.
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