The Function And Mechanism Of Transcription Factor MaAP2a-1 And MabHLH6 In Regulation Of Postharvest Banana Starch Degradation

Banana is a typical climacteric fruit,and bananas accumulate large amounts of starch during fruit development.While the starch rapidly degradesto soluble sugars and the fruit softens after the onset of ethylene release during the postharvest fruit ripening.The degradation of stored starch supports the energy basis for postharvest ripening.Although starch degradation has been well studied in model systems such as Arabidopsis leaves and cereal seeds,theprocesses in starchy fruits during ripening,especially in banana fruit,are largely unknown.In Arabidopsis thaliana,actions of glucan phosphorylation by GWD1 and PWD,glucan can be partially hydrolyzedby hydrolases,followed by phosphoglucan dephosphorylation with SEX4 and LSF1/2,glucan completelydegrades and product is export out of plastid.In this study,according to the model of Arabidopsis,we isolated and characterized 38 genes(?-glucan water dikinases(MaGWD1,MaGWD2,and MaPWD1),phosphoglucan phosphatases(MaSEX4,MaLSF1,and MaLSF2),?-amylase(MaBAM1-MaBAM11),?-amylases(MaAMY2A,MaAMY2 B,MaAMY2C,MaAMY3,MaAMY3 A,MaAMY3B,and MaAMY3C),starch debranching enzymes(MaISA1-MaISA3),?-glucan phosphorylases(MaPHS1 and MaPHS2),disproportionating enzymes(MaDPE1 and MaDPE2),maltose excess proteins(MaMEX1 and MaMEX2),and plastidic glucose transporters(MapGlcT1,MapGlcT2-1,MapGlcT2-2,MapGlcT4-1,and MapGlcT4-2)encoding starch breakdown-related enzymes from banana genome.We analyse theexpression patterns of those genes in pulp during banana fruit ripening and uncovered the action of starch degradation and regulation mechanism of key transcription factor MaAP2a-1 and MabHLH6.The results are as follows.1.The decrease in starch content and increase in total soluble sugars are in good agreement with fruit softening and ethylene production during banana ripening.Moreover,SEM analysis indicated clear change in starch granules morphology from oval with smooth surface to elongated with parallel grooves,indicating the enzymatic effects caused by fruit ripening.2.We isolated and characterized 38 genes encoding starch breakdown-related enzymes from banana genome.Expression analysis revealed that 27 candidate genes(MaGWD1,MaPWD1,MaSEX4,MaLSF1,MaLSF2,MaBAM1-MaBAM4,MaBAM6-MaBAM8,MaBAM10,MaAMY2 B,MaAMY2C,MaAMY3,MaAMY3 A,Ma AMY3 C,MaISA2,MaISA3,MaPHS2,MaMEX1,MaMEX2,MapGlcT2-1,MapGlcT2-2,MapGlcT4-1 and MapGlcT4-2)were significantly induced during banana fruit ripening,with concomitant conversion of starch-to-sugars.While transcripts of MaGWD2,MaAMY2 A,MaBAM9,MaPHS1,MaDPE1,MaDPE2 and MapGlcT1 were decreased and 3 genes(MaAMY3B,MaBAM5 and MaBAM11)had irregular changes and one gene(MaISA1)displayed no obvious variation.3.iTRAQ-based proteomics experiments identified 18 starch degradation-associated enzymes bound to the surface of starch granules,of which 10 proteins(MaGWD1,MaPWD1,MaSEX4,MaLSF1,MaBAM4,MaBAM7,MaAMY2 B,MaAMY2C,MaAMY3,and MaISA3)were markedly up-regulated during ripening.Furthermore,immuno-blotting analyses of starch-related proteins showed that MaGWD1 was present on the surface of starch granules and increased its content in the pulp of ripe bananas.Moreover,ethylene induced the activity of MaGWD1 promoter,indicating that MaGWD1 may play an important role in ethylene-mediated starch breakdown.4.Novel AP2 and bHLH transcription factor,MaAP2a-1 and MabHLH6,were identified based on a yeast one-hybrid screening using MaGWD1 promoter as a bait.Transcript and protein levels of MaAP2a-1 were also decreased during fruit ripening,while MabHLH6 was quite the reverse.Tobacco transient expression experiments confirmed that MaAP2a-1 was a repressor with a transcription repression domain EAR and MabHLH6 was an activator.Futhermore,ethylene can also induced the activity of MabHLH6 promoter.5.Electrophoretic mobility shift assays,chromatin immunoprecipitation and transient expression experiments found that MaAP2a-1 repressed promoters of 15 starch degradation-related genes(MaGWD1,MaPWD1,MaSEX4,MaLSF1,MaBAM1-MaBAM3,MaAMY2 B,MaAMY2C,MaAMY3 A,MaAMY3C,MaMEX1,MaMEX2,MapGlcT2-1and MapGlcT2-2)via binding to the GCC-box or AT-rich motif of their promoters.Moreover,MabHLH6 activated the promoters of 11 starch degradation-related genes(MaGWD1,MaLSF2,MaBAM1,MaBAM2,MaBAM8,MaBAM10,MaAMY3,MaAMY3 C,MaISA2,MaISA3,and MapGlcT2-2 by recognizing their E-box(CANNTG)motifs present in the promoters.