Multiple Forms of Pectinesterase are Present in Tomato and Other Fruit

Tomato

Animation of a ripening tomato

Figure 3. PE isoform profile for enzyme extracted from 10g normal and PE2 antisense tomatoes (isoforms separated using cation exchange on CM sepharose).

Figure 4. PE isoform profile for enzyme extracted from 10g normal and PE2 antisense tomatoes (isoforms separated using affinity chromatography on heparin).

The dominant Pectinesterase isoform in tomato is PE2 (Graph 3, fractions 20-35). This enzyme accounts for greater than 90% of total fruit PE activity. PE2 antisense plants have no detectable levels of PE2, but the minor isoforms of PE1 and PE3 are still detectable (fractions 40-55). Due to their similar properties, PE1 and PE3 are difficult to separate from one another. Our recent work has attempted to separate these isoforms using HRLC with affinity chromatography on Heparin-Sepharose. As with the CM-Sepharose method, two Biorad Econo-Pac columns were connected in series. Preliminary work has indicated that this column may be able to resolve PE1 & PE3 into distinct peaks (Graph 4).

The failure to downregulate the minor isoforms in PE2 antisense plants is due to there being little (if any) similarity between the enzymes at the DNA sequence level. Currently, attempts are being made to characterise these isoforms further. So far, attempts to isolate the gene coding for PE1 have been unsuccessful. A cDNA clone for PE3 has been isolated in our laboratory, and plants have been transformed with a chimeric gene construct to downregulate the enzyme. This should give a clear indication of the role of PE3 in plants, and whether it is involved in fruit ripening.

Multiple Forms of Pectinesterase are Present in Tomato and Other Fruit

Tomato

Further Reading