| Apple（Malus×domestica）is an important economic fruit in China.Its cultivation area and yield in China have long been the largest in the world.Apple fruit color is an important trait to attract costumer attention.Apple mainly has three colours,red,yellow and green.The red colour is caused by anthocyanin accumulation.Because anthocyanin can not only improve fruit appearance,but also provide good benefits for human health,red apples have been deeply loved by consumers.Many studies have deeply analyzed the mechanism of different factors affecting anthocyanin synthesis,but there are still some phenomena that cannot be explained by the existing results.Based on previous studies,this study clarified the function and molecular mechanisms of miR172 and MdMYB28 regulating anthocyanin accumulation in apple fruit skin,providing a theoretical basis for breeding new apple varieties with good color,high quality and healthy functions,thus promoting the sustainable development of the apple industry.The main results are as follows:1.Previous results showed that the content of anthocyanin in fruit skin of miR172 over-expression（miR172OX）apples was decreased.Sucrose-induced anthocyanin biosynthesis was not observed in the leaves of three independent miR1720X lines tested when sucrose concentration of the media was elevated to 7%.In greenhouse conditions,red coloration was not observed on young stems of four independent transgenic plants.These results suggest that over-expression of miR172 reduced red colouration in apple.2.The apple genome contains eight AP2-like genes with miR172 target sequences.A phenology analysis revealed that MdAP21a（MD15G1286400）was most closely related to Arabidopsis AP2 and was expressed at a relatively high level.3.RT-PCR analyses showed that miR172 level was negatively but MdAP2 la mRNA level was positively correlated with fruit skin red colouration during the onsite of anthocyanin biosynthesis in apple fruit skin.4.In Arabidopsis,over-expression of miR172 also reduced anthocyanin accumulation in plantlets cultured on medium containing high concentration（7%）sucrose.We found that red colouration under the miR172 over-expression condition could be partially restored by the MdmAP21a but not the MdAP21a transgene in plantlets cultured on medium containing high concentration（7%）sucrose.5.Over-expression of MdAP21a in transgenic tobacco plants can enhance petal anthocyanin accumulation.Fruit anthocyanin accumulation and red coloration are reduced by downregulating expression of MdAP21a in apple and PavAP2 in cherry using VIGS（Virus-induced-gene-silence）technique.6.Yeast one-hybrid assay indicated that a fragment of MdAP21a protein could directly bind to the promoter of MdMYB10.Luciferase and GUS report gene assays showed that MdAP21a could also activate the promoter of MdMYB10 directly.Yeast-two-hybrid assay indicated that MdAP21a could interact with MdMYB10 protein.7.We analyzed 27 transcriptomes of fruit skin from three apple cultivars ’Huashuo’（red-skinned）,’Hongcuibao’（red-skinned）,and ’Golden Delicious’（yellow-skinned）at 0,2,and 6 days after bag removal.Using pairwise comparisons and weighted gene co-expression network analyses,we constructed 17 co-expression modules.Of them,four modules exhibited significance correlation between MYB10 and UFGT,expression level and anthocyanin content.The genes in the modules are enriched in flavonoid biosynthesis pathways were used to construct gene co-expression network of anthocyanin accumulation.Finally,a R2R3-MYB repressor,MdMYB28,was identified as a key hub gene affecting the different color of fruit skin in apple.8 ’Hongcuibao’,and ’Starking Delicious’ with a high intensity of red colour showed a low level of MdMYB28 expression while ’Huashuo’ and ’Jinxiuhong’ with a low intensity of red colour displayed a high level of MdMYB28 expression.9.MdMYB28 protein was shown to directly bind to the promoter of MdMYB10 in yeast one-hybrid analyses.Over-expression of MdMYB28 decreased the anthocyanin biosynthesis in tobacco flower petals,suggesting that MdMYB28 acts as a negatively regulator of anthocyanin biosynthesis. |
