Plant Molecular Biology Group - Projects


Avocado (Persea americana) Ripening and its Control by Low Oxygen

(A. Kanellis, M. Hassan, K.A. Loulakakis)

In plants, a response to hypoxic and anoxic conditions involves an adaptive mechanism that enables survival in low oxygen stress for several days. Within this context, it is well established that oxygen levels below those in air retard the rate of ripening and softening of climacteric fruits. The mode of action of low oxygen in delaying fruit ripening is not clear. In view of the above evidence and the limited experimental data concerning the mode of action of low oxygen on fruit ripening, we have initiated studies aimed at investigating the biochemical and molecular aspects of low oxygen action on fruit ripening. Our previous results have shown that transferring initiated avocado fruits to 2.5% oxygen for 6 days suppressed the activity, immunoreactive protein and abundance of mRNA of cellulase. The above treatment also produced an alternation in the profile of avocado total proteins which involved suppression, enhancement and induction of new polypeptides. In addition, it has been shown that the range of oxygen levels (2.5-5.5%) which suppressed the appearance of ripening enzymes at the protein and mRNA levels was similar to those oxygen levels that induced the synthesis of new isoenzymes of alcohol dehydrogenase.

In order to distinguish the interactions between oxygen and ethylene, preclimacteric and propylene initiated-ripening avocado fruits were exposed to different low oxygen levels, and the steady state protein pattern was studied by 2-D as well as protein accumulation and gene expression at the level of translatable and accumulated mRNA. The deprivation of oxygen induced the appearance of new polypeptides and mRNA species in both preclimacteric and initiated avocado fruits, whereas it suppressed the synthesis of ripening polypeptides and mRNAs. The synthesis of cellulase was irrespective of the oxygen levels in preclimacteric fruits, whereas it was oxygen dependent, especially between (0-2% O2) in ripening-initiated avocado fruits. Similar results were obtained from northern blot analysis. In low oxygen stressed fruits, induction of alcohol dehydrogenase (ADH) at the protein and mRNA levels took place. Sucrose synthase was induced only in anoxia.

It seems, therefore, that low oxygen exerts its suppressive effect mainly on the de novo synthesised proteins of ripening fruits, whereas it induced the synthesis of new proteins, in order to overcome the stress, irrespective of the developmental stage of the fruit. Th eproject continues with the main objective to isolate low oxygen regulated genes utilizing novel screens in s. cerevisae.

Within the context of avocado ripening, it was also shown that the existence of message heterogeneity in mRNA of avocado cellulase, reported by others, was also reflected in a heterogeneity of cellulase isoenzymes during ripening. It was demonstrated that cellulase occurred in multiple active forms in crude protein extracts based on native IEF gels and visualized by activity staining and immunodetection.

Another separate activity addressed the regulation of glutamate dehydrogenase (GDH) and glutamine synthetase, two nitrogen-metabolizing enzymes, during the development and ripening of avocado fruit. Interestingly, there was a shift in the isoenzymic profile of GDH during ripening, with a predominance of the more cathodal isoenzymes in the unripe fruit and in most of the anodal isoenzymes in ripe fruit. 2-D analysis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the over-ripe fruit was due to the accumulation of the -polypeptide. It should be noted that the physiological role of the induction of GDH during avocado ripening is not known.

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