Global analysis of gene expression during development and ripening of citrus fruit flesh. A proposed mechanism for citric acid utilization

Abstract

Microarrays of cDNA have been used to examine expression changes of 7000 genes during development and ripening of the fruit flesh of self-incompatible Citrus clementina, a non-climateric species. The data indicated that 2243 putative unigenes showed significant expression changes. Functional classification revealed that genes encoding for regulatory proteins were significantly overrepresented in the up-regulated gene clusters. The transcriptomic study together with the analyses of selected metabolites highlighted key physiological processes occurring during citrus fruit development and ripening such as water accumulation, carbohydrate build-up, acid reduction, pigment substitutions (carotenoid accumulation and chlorophyll decreases) and ascorbic acid diminution. Often, the combined analyses strongly suggested prevalence of specific metabolic alternatives. This observation has been exemplified with the proposal for a mechanism for citrate utilization, a process of much importance in citrus industry. Microarray data validated by real-time RT-PCR suggested that citrate was sequentially metabolyzed to isocitrate, 2-oxoglutarate and glutamate. Thereafter, glutamate was both utilized for glutamine production and catabolyzed through the gamma-aminobutirate (GABA) shunt (GABA -> succinate semialdehyde -> succinate). This last observation appears to be of special relevance since it links the proton consuming reaction glutamate + H+ -> GABA + CO2 with high acid levels. GG-MS determinations showed that glutamate was constant while GABA levels decreased at ripening in agreement with a feasible activation of the GABA shunt during acid catabolism. This suggestion provides a convincing explanation for the strong reduction of both citrate and cytoplasmatic acidity that takes place in citrus fruit flesh during development and ripening.