Mechanisms Of B-box Transcription Factor Mediated UV-B And Temperature Regulation On Apple Fruit Coloring

Apple color is an important agronomic trait that determines the market value of apples.Anthocyanins are the main pigments that determine the color of apple fruits.Their physiological synthesis pathways are affected by many external factors,such as light,temperature,hormones and so on,especially Ultraviolet-B(UV-B)and temperature.UV-B promotes anthocyanin synthesis by stimulating the expression of key genes in anthocyanin synthesis pathway.Low temperature increased the photosensitivity of apple fruits,and then promoted the accumulation of anthocyanin in apple fruits,and protected plants from oxidization and low temperature injury.High temperature inhibited the synthesis and accumulation of anthocyanins.UV-B and temperature as signaling regulating anthocyanin synthesis,and many key transcription factors in their pathways have been identified.However,the mechanism of interaction between UV-B and temperature as signaling regulating anthocyanin synthesis in fruit trees remains unclear.B-box(BBX)proteins are a kind of zinc finger structure transcription factor,which contain one or two B-box domains.BBX proteins play an important role in the regulation network of plant growth and development.In recent years,the function of BBX proteins have been studied more and more deeply.Especially in light morphogenesis,flowering control and response to stress.The fourth subfamily of BBX transcription factors has eight members(BBX18-BBX25).Their roles in plant photomorphogenesis have been extensively studied.BBX21 and BBX22 positively regulate photomorphogenesis,BBX24 negatively regulates photomorphogenesis.However,their studies in the UV-B and temperature signaling pathways are relatively rare,and the mechanism of their regulation of anthocyanin synthesis is still unclear.In order to further explore the functions of BBX transcription factors and their roles in UV-B and temperature signaling pathways.In this study,the ‘Yanfu 3’ was used as material.Studies were carried out on screening of expression differences,cloning of MdBBX transcription factor genes,validation and mining of gene functions.The main results are as follows:1.The test sheds were set up in the orchard to deal with the de-bagged fruits by UV-B treatment.The quantitative real-time expression of BBX transcription factor IV subfamily were analyzed to screen the differences.RT-qPCR results showed that the expression of MdBBX20 was significantly up-regulated under UV-B conditions,while MdBBX24 was down-regulated.In this study,the differentially expressed MdBBX20 and MdBBX24 were used as target genes for further functional verification and mining.2.MdBBX20 and MdBBX24 were overexpressed in apple ‘orin’ callus and red callus respectively,and then cultured under UV-B conditions.The results showed that compared with wild type callus,the apple ‘orin’ callus overexpressed MdBBX20 turned red and promoted the synthesis of anthocyanin and the expression of structural genes MdCHI,MdDFR and MANS.Overexpression of MdBBX24 resulted in yellowing of red callus,inhibiting anthocyanin accumulation and inhibiting the expression of structural genes MdDFR,MdANS and MdUFGT.Therefore,MdBBX20 can promote anthocyanin accumulation in response to UV-B,while MdBBX24 inhibits anthocyanin synthesis under UV-B conditions.3.Through yeast two-hybrid experiment,bimolecular fluorescence complementation experiment and Pull down assay in vitro and in vivo,respectively,it was verified that MdBBX20 and MdBBX24 could interact with MdHY5 to form protein complexes.By decomposing the domain,the second B-box domain of MdBBX20/24 and the b-ZIP domain of MdHY5 are necessary for their interaction.The results of LUC fluorescence reporter assay showed that MdBBX20 can promote the binding of MdHY5 to MdMYB1 promoter,while MdBBX24 has the opposite effect.4.Through yeast one-hybrid experiment,EMSA experiment and CHIP assay,it was confirmed that MdBBX20 can specifically recognize and bind to G-box in the promoter of MdDFR and MdANS,and promotes their expressione.By yeast onehybrid experiment,EMSA and LUC reporter assay,MdBBX24 can bind to G-box in the promoter of MdANS and MdUFGT,and inhibits their expression.Meanwhile,LUC reporter assay also showed that MdBBX24 inhibited the binding of MdHY5 to downstream structural gene promoter.5.The cis-acting elements in MdBBX20 promoter were analyzed.It was found that there was a low temperature response element LTR in MdBBX20 promoter.To explore the role of MdBBX20 in low temperature signaling pathway.The results showed that the expression of MdBBX20 was significantly higher under low temperatue condition.At the same time,‘orin’ callus overexpressed MdBBX20 and wild-type callus were cultured under different UV-B and temperature conditions.It was found that ‘orin’ callus overexpressed MdBBX20 accumulated the most anthocyanin under UV-B/low temperature conditions.Interestingly,even without UVB,‘orin’ callus overexpressed MdBBX20 could accumulate anthocyanin only under low temperature conditions.This indicates that MdBBX20 can promote the synthesis of anthocyanin in response to low temperature.6.Since MdBBX20 can respond to low temperature and has a low temperature response element in the promoter.The results of yeast one-hybrid experiment and EMSA experiment showed that MdbHLH3,an important transcription factor in the low temperature response pathway,could specifically recognize and bind to LTR cisacting elements in the MdBBX20 promoter.The expression of MdbHLH3 was also significantly increased at low temperature.These indicate that MdBBX20 can promote anthocyanin synthesis in response to low temperature with the participation of MdbHLH3.7.The cis-acting elements in the promoter of MdBBX24 were analyzed.It was found that there was a heat shock response element HSE in the promoter of MdBBX24.Similarly,to verify the relationship between MdBBX24 and high temperature signal.The de-bagged apples were cultured in light incubators under different temperatures and UV-B conditions.The results showed that the expression of MdBBX24 was down-regulated under UV-B conditions and up-regulated under high temperature conditions,but the anthocyanin content in apple peel and the expression of anthocyanin synthesis structural genes were decreased.Meanwhile,yeast one-hybrid experiment and EMSA experiments showed that heat shock transcription factor MdHSF3b/4a could specifically recognize and bind to HSE cis-acting elements in MdBBX24 promoter.The results of LUC fluorescence reporter assay showed that MdHSF3b/4a could promote the expression of MdBBX24.This indicated that MdBBX24 could inhibit the accumulation of anthocyanin and the color of apple peel in response to high temperature with the participation of MdHSF3b/4a.This study demonstrates that MdBBX20 can respond to both UV-B and low temperature.The interaction between MdBBX20 and MdHY5 clarifies for the mechanism of regulating anthocyanin synthesis under the interaction of UV-B and low temperature signals.It also indicated that the expression of MdBBX24 was inhibited by UV-B and responded to high temperature to inhibit anthocyanin synthesis.