| Dwarfing dense planting is the development trend of apple industry in the world.The main way to achieve dwarfing dense planting is to use dwarfing rootstock.The growth and development of trees is a very complex biological process,and the relationship between various factors is complex.Although dwarf rootstocks are widely used in production,the dwarfing mechanism and its regulation mechanism of apple are still unclear.BARLEY B RECOMBINANT / BASIC PENTACYSTEINE(BBR / BPC)transcription factor is a small plant-specific transcription factor family,and it has been found that BPC transcription factors can induce plant dwarfing.However,how BPC regulates plant structure is an open question.We found that MdBPC2,a transcription factor in the BBR / BPC family,is highly expressed in dwarf rootstocks in apples.In order to explore its relationship with dwarfing,this paper identified and verified the expression of MdBPC2 regulating auxin biosynthesis genes in apples based on RNA-Seq and DAP-seq analysis,and proved its mechanism and function in dwarfing.The molecular regulation mechanism of apple dwarfing was analyzed.The main results are as follows:1.MdBPC2 acts as a transcriptional suppressor to negatively regulate auxin accumulation.Six MdBPC genes were identified from apple genome by bioinformatics method.Based on RT-q PCR analysis,it was found that Class I BPC transcription factor MdBPC2 was highly expressed in apple dwarfing rootstocks,and tissue-specific expression analysis showed that it had the highest expression in apple stem tips.Subcellular localization results showed that MdBPC2 protein is located in the nucleus,and its transcriptional selfactivation activity in yeast and transcriptional function analysis confirmed that MdBPC2 protein is a transcriptional suppressor.To further investigate its function,overexpressed and silenced MdBPC2 transgenic apple lines were obtained.Overexpression of MdBPC2 significantly reduced plant height,changed leaf morphology,and limited root growth.Plant hormone measurements showed that the auxin content of MdBPC2 overexpressed transgenic plants decreased significantly.Exogenous auxin supplementation restored the height,leaf morphology and root development of MdBPC2 overexpressed transgenic apple plants.Treatment with auxin inhibitors(p-chlorophenoxyisobutyric acid,PCIB)inhibited auxin accumulation in wild-type plants,resulting in phenotypes similar to those of MdBPC2 overexpressed transgenic apple plants.These results suggest that MdBPC2,which has transcriptional inhibitory function,plays an important role in mediating auxin accumulation.2.MdBPC2 inhibits the expression of auxin biosynthesis genes MdYUC2 a and MdYUC6 b.RNA-seq data analysis showed that MdBPC2 inhibited the expression of several auxin synthesis genes MdYUC2 a,MdYUC6 b and MdGH3.1a.DAP-Seq data and electrophoretic mobility shift assay(EMSA)results showed that MdBPC2 regulates the expression of target genes by binding to GAGA-Rich elements.Analysis of several auxin synthesis gene promoters revealed that MdBPC2 may regulate the expression of MdYUC2 a and MdYUC6 b.Yeast single hybridization(Y1H)and chromatin immunoprecipitation(CHIP-q PCR)confirmed that MdBPC2 directly binds to GAGA-Rich elements in the promoters of MdYUC2 a and MdYUC6 b.Dual luciferase analysis and RT-q PCR confirmed that MdBPC2 inhibited the expression of MdYUC2 a and MdYUC6 b.These results indicate that MdBPC2 negatively regulates the expression levels of MdYUC2 a and MdYUC6 b and inhibits the accumulation of auxin.3.The MdBPC2 protein recruits the Pc G protein MdLHP1 to the MdYUC2 a and MdYUC6 b loci,and subsequently catalyzed the inhibitory histone labeling H3K27me3 to inhibit the expression of MdYUC2 a and MdYUC6 b.CHIP-q PCR results showed that H3K27me3 modification levels of MdYUC2 a and MdYUC6 b loci were significantly increased after MdBPC2 overexpression.It was found that MdBPC2 may interact with Pc G protein MdLHP1 by using yeast two-hybrid sieve library experiment.The interaction between MdBPC2 and MdLHP1a/b in vitro and in vivo was confirmed by Bimolecular Fluorescent Complimentary(Bi Fc),Luciferase Complementation Assay(LCA)and PullDown experiments.When MdLHP1-Flag fusion protein was expressed in wild-type and transgenic plants,the enrichment level of MdLHP1-Flag at MdYUC2 a and MdYUC6 b sites was significantly increased in plants overexpressing MdBPC2.However,the H3K27me3 levels of MdYUC2 a and MdYUC6 b loci decreased significantly after the expression of MdLHP1 was silenced in overexpressed transgenic lines.Meanwhile,in apple callus,expression of LUC genes driven by MdYUC2 a and MdYUC6 b promoters containing mutated GAGA-Rich elements significantly reduced H3K27me3 levels and removed LUC gene inhibition.These results indicated that MdLHP1,after enrichment in MdYUC2 a and MdYUC6 b loci,catalyzed H3K27me3 to inhibit their expression.In this process,the binding of MdBPC2 on MdYUC2 a and MdYUC6 b promoters is a necessary condition.4.MdLHP1-mediated H3K27me3 inhibition of MdYUC2 a and MdYUC6 b directly affects auxin content.In order to further explore the direct effect of the interaction between MdBPC2 and MdLHP1 on auxin content,transgenic apple callus overexpressing MdBPC2 were obtained.RT-q PCR analysis showed that the overexpression of MdBPC2 inhibited the transcription of MdYUC2 a and MdYUC6 b,which was consistent with the results of transgenic plants.On the basis of overexpression of MdBPC2,after silencing MdLHP1 gene,the auxin content of transgenic callus was detected by DR5: GUS reporter gene.The results showed that the overexpression of MdBPC2 inhibited the accumulation of auxin,resulting in decreased GUS activity.On the contrary,this inhibition was eliminated after silencing MdLHP1 gene.GUS activity was even higher than that of wild type.These results suggest that MdLHP1 is involved in the regulation of auxin biosynthesis by MdBPC2 during growth and development. |
