A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening

Abstract

Although starch degradation has been well studied in model systems such as Arabidopsis leaves and cereal seeds, this process in starchy fruits during ripening, especially in bananas, is largely unknown. In this study, 38 genes encoding starch degradation-related proteins were identified and characterized from banana fruit. Expression analysis revealed that 27 candidate genes were significantly induced during banana fruit ripening, with concomitant conversion of starch-to-sugars. Furthermore, iTRAQ-based proteomics experiments identified 18 starch degradation-associated enzymes bound to the surface of starch granules, of which 10 were markedly up-regulated during ripening. More importantly, a novel bHLH transcription factor, MabHLH6, was identified based on a yeast one-hybrid screening using MaGWD1 promoter as a bait. Transcript and protein levels of MabHLH6 were also increased during fruit ripening. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transient expression experiments confirmed that MabHLH6 activates the promoters of 11 starch degradation-related genes, including MaGWD1, MaLSF2, MaBAM1, MaBAM2, MaBAM8, MaBAM10, MaAMY3, MaAMY3C, MaISA2, MaISA3 and MapGlcT2-2 by recognizing their E-box (CANNTG) motifs present in the promoters. Collectively, these findings suggest that starch degradation during banana fruit ripening may be attributed to the complex actions of numerous enzymes related to starch breakdown at transcriptional and translational levels, and that MabHLH6 may act as a positive regulator of this process via direct activation of a series of starch degradation-related genes.

Keywords: bHLH; banana fruit; iTRAQ; starch degradation; transcriptional activator.