Regulation of polygalacturonase gene expression during tomato fruit ripening
Abstract
Changes in gene expression during tomato (Lycopersicon esculentum Mill.) pericarp development and ripening, including the softening enzyme polygalacturonase (PG), were quantified using the normally ripening cultivar Rutgers and compared to changes exhibited in nonripening mutants (rin, nor, Nr, Gr, Long Keeper) and their heterozygotes with Rutgers. The relative predominance of many proteins and mRNAs changed during normal pericarp development and ripening, but several proteins and mRNAs were absent or reduced in homozygous rin, nor, and Nr. PG2, an isoform of PG, was purified and used for polyclonal antibody production. Biochemical characterization revealed that PG2 (1) is synthesized on bound polyribosomes as a 54 kD precursor, (2) is composed of 44 kD (PG2A) and 45 kD (PG2B) enzymatically active isoforms containing 3 to 4 kD of carbohydrate, and (3) shows a 23-fold higher Km for polygalacturonic acid of 1 kD versus 31 kD, yet only exhibits a two-fold increase in V$\sb{\rm max}$ over this range. Molecular cloning of PG was accomplished by constructing and immunoscreening a $\lambda$gt11 cDNA library representing Rutgers turning stage poly (A)$\sp+$ RNAs. PG mRNA, protein, and enzyme activity peaked at the turning, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. No PG gene expression was found in Rutgers preclimacteric fruits or vegetative tissues, while expression was low (0 to 15% of normal) in pericarp from all five mutants. Maximum expression of the PG gene in pericarp from heterozygotes (rin, nor, Nr, Gr, and Long Keeper crossed with Rutgers) at the mRNA level was 25, 13, 17, 5, and 62%, at the protein level was 166, 110, 15, 6, and 104%, and at the enzyme activity level was 69, 37, 4, 1, and 50% of maximum levels found in Rutgers, respectively. Transcriptional regulation of PG mRNA accumulation and other post-transcriptional mechanisms resulting in delayed PG protein and enzyme activity accumulation were evident. While extensive restriction fragment length polymorphisms of the PG gene were observed in different Lycopersicon and Solanaceous species, no loss of or detectable rearrangements in PG gene sequences were evident in the mutants or heterozygotes.
Degree
Ph.D.
Advisors
Handa, Purdue University.
Subject Area
Molecular biology
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