AG-WUS-LHP1-lncRNA Regulate Multiple Pistil Formation And Development In Japanese Apricot

Japanese apricot(Prunus mume Sieb.et Zucc.),an essential member of Rosaceae,is a fruit and ornamental crop.Japanese apricot is one of the most precious processed fruit with great economic importance used as preserved and in alcoholic beverage industries.Generally,in Japanese apricot,a perfect flower retains a normal pistil development that leads towards a single fruit formation.But,Japanese apricot also own a few cultivars with two or more pistils,which lead to multiple fruit formation while others sterility result decreases the yield and low fruit quality.However,there are few reports on the multiple pistil studies in Prunus mume,and the molecular mechanism of multiple pistil formation is quite unknown.Therefore,in this study we selected Japanese apricot single pistil cv'Qingjia No.2'(QJN2)and multiple pistils cv 'Da Yu'(DY)as the research plant materials.The key period of multiple pistils differentiation was determined through paraffin section and explore the relationship between the spatiotemporal expression of AG and WUS with multiple pistil differentiation in Prunus mume.The sites and amount of AG and H3K27me3 bound to WUS locus were determined.Furthermore,we also determined the differential expression of IncRNAs and role of polycomb protein(PcG)member LHP1 in flower development process in QJN2 and DY.Finally,we proposed the model of AG-WUS-LHP1-lncRNA mutual regulation pistil development in Japanese apricot.The specific research contents and conclusions are as follows:1.In this study,we statistics pistil quantity,observed the pistil differentiation process and related physiological indexes of single pistil cv QJN2 and multiple pistils cv DY in Japanese apricot.According to the statistics of the number of pistil,all QJN2 organs are single pistils,while DY always own two or three pistils.The similar surface cell shape of two types of pistil was observed by SEM.The differentiation process result showed that the same pistil differentiation process was observed in the DY and QJN2.In the process of pistil differentiation of DY,the pistil primordial was in pre-differentiation stage at the late September,but the sepals and petals have appeared.In early October,the pistil primordial entered in early differentiation stage and started to expand.From late October to late November,the pistil primordial was in the differentiation stage.The early October is the key stage for pistil differentiation and determines the number of pistils.The content of IAA and ZT in DY was significantly higher than that of QJN2 in the early differentiation stage,which might effect the multiple pistil formation.2.AG and WUS are the two important genes play a significant role in flower organ development.In pistil pre-differentiation stage of QJN2 and DY,AG and WUS gene expression showed a negative relationship that might be related to the negative regulation between them.Besides,the AG gene always showed a higher expression in the pistil differentiation stage and late differentiation stage,which suggested that AG might be associated with the ovule and other floral organ development.By using plants ChIP method with optimization,we determined the ultrasonic conditions 16%power,3 second for each operation,9 second for each interval,and 3 minutes for the total time,is suitable for our study.Western blot experiments confirmed AG and H3K27me3 proteins exist in both QJN2 and DY.The ChIP-qPCR results showed that the H3K27me3 bound to the WUS locus,and the binding regions of WUS 1,WUS 2 and WUS 3 were significantly higher in QJN2 than DY.Moreover,the binding of QJN2 to AG protein at the WUS 2 region was significantly higher than that of DY.These results suggesting that H3K27me3 is the important way to inhibit the expression of WUS,and the process of AG inhibiting the expression of WUS might be regulated by H3K27me3.3.Long non-coding RNAs(lncRNAs)are the transcripts more than 200 bp in length do not encode proteins.Up to the present,it has been reported that lncRNAs play an essential role in developmental processes through their regulatory functions.However,their characteristics,expression inheritance patterns and functions in Japanese apricot are quite unidentified.In this study,we investigated a complete pistil development lncRNA profiles between single pistil cv QJN2 and multiple pistils cv DY through RNA-seq in Japanese apricot.2572 unique lncRNAs and 24648 genes mapped to Japanese apricot genome,moreover,591 novel lncRNAs were also predicted.Both unique and novel lncRNAs are shorter in length than the mRNAs,and the overall expression level of lncRNAs was lower than mRNAs in Japanese apricot.186 unique lncRNAs,1638 genes and 89 novel lncRNAs were identified as significant differential expressed in QJN2 compared with DY.We predicted 421 target genes of DEULs and 254 target genes of DENLs.153 miRNAs were predicted interacted with 100 DEULs while 112 miRNAs were predicted interacted with 55 DENLs.LncRNA XR₅14690.2 was predicted to target ppe-miR172d and the expression of XR₅14690.2,ppe-miR172d and AP2 revealed that XR₅14690.2 down-regulated ppe-miR172d and up-regulated AP2.Down-regulated DENLs TCONS₀0032517,hampered induction of A-ARR genes by targeting with ppe-miR160a/b in DY.A-ARR negatively regulated cytokinin signaling.ZT treatment significantly promoted AP2 expression in flower buds.This study showed the first characterization of lncRNAs involved in pistil development and provides new indications to elucidate how lncRNAs and their targets play a role in pistil differentiation and flower development in Japanese apricot.4.LHP1 is an important member of PRC1 and plays an important role in flower development.According to the analysis of LHP1 protein,we found that LHP1 is hydrophilic protein contains 450 amino acids with 50.21 kDa molecular weight,5.24 theoretical isoelectric point,and LHP1 is predicted to be located in the nucleus.By comparison with the CDD database,we found that LHP1 protein contained CD and CSD domain.These two domains indicate that LHP1 has the ability to recognize H3K27me3 modification label and bind proteins in Japanese apricot.The results of CoIP LC-MS/MS showed that LHP1 could interact with proteins.In QJN2,LHP1 interacts with histone H3.2.Histone H3.2 can formation H3K27me3.Western blot shown the QJN2 own higher level of LHP1 binding to H3K27me3 than DY,which is conducive to regulating the expression of related genes,e.g.WUS.In addition,LHP1 interacted with FLK in both samples,which would affect flower development by regulating flower organ identification genes.In our study,the results of LHP1 interact with protein provides a basis for further study on the function of LHP 1 in Japanese apricot.