Molecular cloning and expression of pectin methylesterase genes in Lycopersicon esculentum (tomato)

Robert Woodrow Harriman, Purdue University

Abstract

Pectin methylesterase (PME) is a ubiquitous plant enzyme which demethoxylates pectin resulting in the formation of free carboxyl groups which may form Ca$\sp{++}$ cross-bridges. While PME has been found in every plant examined, it is a particularly abundant protein in developing tomato fruit. Deesterification of pectin by PME and depolymerization by polygalacturonase are proposed to be involved in fruit softening during tomato fruit ripening. To study the expression of PME in developing tomato fruit, PME was purified from the pericarp of mature green (MG) tomato fruit, anti-PME antibodies were raised in chickens and a cDNA clone was isolated from a $\lambda$gt11 expression library constructed with pericarp MG poly A$\sp{+}$ RNA. Anti-PME antibodies and a PME cDNA clone were used to study PME's expression at the protein, specific activity, and mRNA levels. A gradual increase in PME activity begins at day 20 and continues until the MG stage when a 2-fold increase occurs between the MG and breaker (Br) stages. Only low levels of PME activity are detected in 10 and 15 day old fruit. The increase in activity parallels an increase in PME protein, however the level of PME protein increases beyond the Br stage while PME activity begins to decline. Even though PME activity levels comparable to 25 day old fruit were found in root tissue, no detectable PME protein was observed using Western blot analysis. PME mRNA was first detected in 15 day old fruit and reached a steady-state maximum by 30 DAF. Elevated levels of PME mRNA were maintained through the MG stage and began decreasing as the fruit ripened. No detectable PME mRNA was observed in root, stem, leaf and 10 day old fruit. Localization of PME and PG mRNA in developing tomato fruit using tissue-printing demonstrated PME and PG mRNA are expressed in the same tissue, however PME mRNA was detectable in 15 day old fruit, while PG mRNA first appeared in Br fruit. Genomic analysis of PME gene structure was aided by the isolation of two genomic DNA clones ('VFNT Cherry'). Partial sequencing demonstrated the existence of three tandemly repeated PME genes. Southern blotting for PME genes of genomic DNA isolated from the cultivars 'VFNT Cherry' and 'Rutgers' showed considerable restriction fragment length polymorphism. Two introns have been located within the coding region of the PME genes studied.

Degree

Ph.D.

Advisors

Handa, Purdue University.

Subject Area

Botany|Molecular biology

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