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returning the fruits to higher ripening temperatures (Chan et al., 1985; Lyons &
Breidenbach, 1987).
Pectinmethylesterase (PME) activity has been reported to increase during the
development of banana (Brady, 1976), apple (Knee, 1978), avocado (Awad et al., 1979)
and papaya (Paul & Chen., 1983) fruits. The exact role of PME in
Carica papaya
fruit
development and ripening is yet to be determined. However, it has been hypothesised that
destherefication of pectin by PME and further depolymerisation by polygalacturonase (PG)
are involved in fruit softening. This hypothesis is based on the observation that
demethylation of pectin by PE causes a several fold increase in cell wall solubilisation by
polygalacturonase (Pressey and Avants, 1982). PME, in addition to other pectolytic
enzymes, has been implicated in fruit ripening (Basic et al., 1988). This cell wall
metabolising enzyme is responsible for the demethylation of galacturonic acid residues in
high molecular weight pectin, each methyl group being converted to a proton and methanol
(Hall et al. 1993). According to Ali et al. (1993), PG, PME and E-galactosidade may
collectively play significant roles in the development of the chilling injury symptom of
increased - susceptibility - to disease commonly observed in papayas upon returning chill-
stored fruits to warmer environments.
The aim of this study was to investigate the significance of PME to differential
softening and to characterise the PME expression during papaya fruit development and
ripening at the biochemical level and molecular mRNA translation.
1. Material and Methods
1.1 Plant material and sampling
After harvest, mature green papayas were brought from Guinea-Bissau to the Laboratory
and were allowed to ripe at 25
o
C. The fruits were sampled at different ripening stages (1, 3,
5, 7, 9 and 11 days), cut transversally in two parts and seeds were removed. Inner mesocarp
was separated from the outer mesocarp and was homogenised each in liquid nitrogen using
warring blander. The homogenised pulp was instantly frozen at -80
o
C.
1.2 RNA isolation
The tissues were ground to a fine powder in liquid nitrogen using a warring blender. Using
a metal spatula, chilled in liquid nitrogen, the powder was quickly transferred to tubes
containing mixture (1:1) of extraction buffer (sodium acetate, EDTA and SDS) and phenol
(pH 4.3), preheated at 65
o
C for 5 min. After homogenisation by vortexing for 5 min, ½ x
volume of Chloroform: Isoamyl alcohol (24:1) was added. After vortexing for 5 min, the
homogenate was spined at 10000 rpm for 10 min at 4
o
C. Using a sterile glass pipette, the
upper aqueous phase was transferred to polypropylene tubes and equal volume of
chloroform isoamyl alcohol (24:1) was added. Vortexing for 5 min and centrifugation for
10 min at 10000 rpm at 4
o
C were repeated. Using a sterile glass pipette, the upper aqueous
phase was again transferred to polypropylene tubes and equal volume of chloroform:
isoamyl alcohol was added and vortexed for 5 min. The sample was transferred to Corex
tubes and spined at 10000 rpm for 10 min at 4
o
C. The upper aqueous phase was
transferred to Corex tubes and 1/3 volume of 8 M LiCl was added and precipitation took
place overnight at 4
o
C. A new centrifugation at 10000 rpm for 10 min at 4
o
C. Pellet was
dissolved with 2 M LiCl by vortexing and centrifuged 10 min at 10000 rpm at 4
o
C. The
two previous steps were repeated twice. The pellet was dissolved with 3 M Sodium acetate
