Arabidopsis TT19 functions as a carrier to transport anthocyanin from the cytosol to tonoplasts.
Anthocyanins are synthesized in the cytosolic surface of the endoplasmic reticulum (ER) but dominantly accumulate in the vacuole. Little is known about how anthocyanins are transported from the ER to the vacuole. Here, we provide evidence supporting that Transparent Testa 19 (TT19), a glutathione S-transferase (GST), functions as a carrier to transport cyanidin and/or anthocyanins to the tonoplast. We identified a novel tt19 mutant (tt19-7), which barely accumulates anthocyanins but produces a 36% higher level of flavonol than the wild-type (WT), from ethyl methanesulfonate mutagenized seeds. Expressing TT19-fused green fluorescence protein (GFP) in tt19-7 rescues the mutant phenotype in defective anthocyanin biosynthesis, indicating that TT19-GFP is functional. We further showed that TT19-GFP is localized not only in the cytoplasm and nuclei, but also on the tonoplast. The membrane localization of TT19-GFP was further ascertained by immunoblot analysis. In vitro assay showed that the purified recombinant TT19 increases water solubility of cyanidin (Cya) and cyanidin-3-O-glycoside (C3G). Compared with C3G, Cya can dramatically quench the intrinsic tryptophan fluorescence of TT19 to much lower levels, indicating a higher affinity of TT19 to Cya than to C3G. Isothermal titration calorimetry analysis also confirmed physical interaction between TT19 and C3G. Taken together, our data reveal molecular mechanism underlying TT19-mediated anthocyanin transportation.
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai 200032, China.
Yi
Y
Sun
Hong
H
Li
Ji-Rong
JR
Huang
D000483:Alleles; D000872:Anthocyanins / Q000378:metabolism*; D017360:Arabidopsis / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D044948:Flavonols / Q000096:biosynthesis; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
D000872:Anthocyanins; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D044948:Flavonols; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013395:Sucrose; C017154:cyanidin; D005982:Glutathione Transferase; C484247:TRANSPARENT TESTA 19 protein, Arabidopsis