Molecular Mechanism Of The KNOX And SBP Family Genes On Regulating Stem Apical Development In Citrus

Citrus tree developed shoots at specific stage,the major phenomenon which restrained the apex development and apical dominance released lead withering and falling off shoot is called "self-pruning".This physiological mechanism occurred in several woody plants,but its specific regulatory mechanism is still not recognized.Although,the tip self-pruning is the key component of flowering transformation in Citrus of trifoliate orange.With the completion of self-pruning,most of the shoots of citrus will change from vegetative growth to reproductive growth.In previous studies on self-pruning,members of the KNOX(KNOTTED1-like homeobox)and SBP-box(SQUAMOSA-promoter binding protein)gene family were found to be involved in regulating the self-pruning process of sweet oranges.Based on the published citrus genome,this study isolated and identified these two gene family members.For some gene members closely related to self-pruning,their functions were identified through plant genetic transformation.The CsHD1 gene of KNOX family member may have an important role in regulating the self-pruning process of citrus apex,but its regulatory mechanism is still unclearand,yeast single-hybrid,EMSA,and dual luciferase systems were used to identify the upstream regulatory factor CsERF of the CsHD1 gene;through yeast two-hybrid,dual-fluorescent complementation and some other technology to isolate its downstream target gene or interaction protein CsHD2,and further use genetic transformation to verify the function of its upstream regulatory genes and interaction protein;finally,by applying phytohormone treatment to the shoots of citrus,the change of self-pruning time and related genes during the investigation regarding the expression pattern of hormonal response,the entire network regulation pathway was analyzed,and the molecular mechanism of self-pruning of the shoot tips of citrus was explained,laying an important foundation for the study of the development process of citrus apex.The main findings are as follows:1.Isolation and identification of KNOX family members of citrus and the CsHD1 regulatory network.In the citrus genome,there are nine members of the KNOX family of citrus.The entire family can be divided into two major categories,including six members of class I and three members of class II.The conserved domains are certainly conservative of the entire family members found in all species.Previous studies found that the CsHD1 gene,which is closely related to self-pruning,belongs to the KNOX family of citrus.It has been shown by transgenic verification that it can regulate the development of leaves,the development of stems and apical meristems,and it also has a certain effect on the regulation of flowering and flowering time in plants.Using yeast two-hybrid,we found that CsHD1 can interact with the CsHD2 protein.Functional analysis found that citrus CsHD2 gene inhibited the tip development of transgenic plants and promoted the development of plant lateral buds,indicating that the CsHD2 gene has important regulatory functions in the development of plant tip tissues and lateral buds.Yeast one-hybrid was used to find an ERF transcription factor combined with the GCC-like box element on the CsHD1 promoter to inhibit the expression of CsHDl gene.Functional analysis found that CsERF can promote tissue aging and increase the content of ethylene and reactive oxygen species.2.The CsHD1,CsHD2 and CsERF gene have important regulatory functions in the self-pruning process of citrus shoots.By analysis of the expression profiles of citrus CsHD1 and CsHD2 transgenic tobacco we found that a large number of auxin and ethylene-induced genes showed significant differential expression.CsHD1 transgenic tobacco increased auxin content and ethylene content decreased;CsHD2 transgenic tobacco auxin content decreased,ethylene content Content increased.When citrus was treated with auxin,and ethylene,CsHD1,CsHD2,and CsERF showed a clear response to hormones.Auxin promoted the expression of CsHD1 and inhibited the expression of CsHD1 and CsERF.Ethylene did the opposite.After auxin treatment,the self-pruning of sweet orange is delayed,and ethylene can promote the time of self-pruning.3.Analysis of the regulatory network model of the self-pruning mechanism of the citrus shoot tips.Analysis of the regulatory network of the self-pruning mechanism of the citrus shoot tip tissue found that after the citrus shoot grew to a certain stage,the ethylene content in the apical tissue increased and induced the expression of CsERF.CsERF binds to elements on the CsHD1 promoter directly inhibits the transcriptional expression of CsHDl,Causing the protein content of the complex formed by binding to CsHD2 to decrease,which caused a large accumulation of CsHD2 protein in the apical tissue of the citrus,inhibited the further growth of the citrus apex,and induced the self-shear phenomenon;In contrast,the expression of CsERF at the tip of citrus is inhibited under conditions of high auxin content,the inhibitory effect of CsERF to CsHD1 was released,and eventually the CsHD1 content increased and the CsHD2 content decreased in the citrus apex.At this time,the citrus apex grew normally and the apex advantage continued to be maintained.4.Isolation,identification and bioinformatics analysis of citrus SBP-box family genes.Citrus SBP-box family genes contain 15(CclSBPs)members.Based on phylogenetic analysis and the structure of introns and exons,the 15 CclSBPs members can be divided into six categories;according to the motif structure analysis prediction,the CclSBP family of Clementina shares similarities with the SPL gene family of Populus trichocarpa and Arabidopsis thaliana.In addition,depending on the similarity of the sequences and the conservation of the conserved domains,this family is divided into different types,and their functions are likely to be redundant.In addition,10 of the CclSBPs members contained target sites of microRNA156(CclmiR156).5.Analysis of citrus SBP-box family expression pattern and functional identification of some members.The tissue-specific expression analysis of each member of CclSBPs proved that it has diverse expression patterns.Studies on the dynamic changes in expression patterns of 15 CclSBPs members during flower induction during the late stage of flower development in the apical tissue of lemon apex showed that some CclSBPs can maintain the apical dominance of citrus and are related to the flower induction and flowering process;Through analysis of the co-expressing protein regulatory network analysis,CclSBPs have a variety of complex regulatory network pathways during plant development;in addition,the transgenic function identification of CclSBP6 and CclSBP7 found that members of the SBP-box family of citrus have certain functions for the plant to regulate flowering time and to maintain the normal development of apical tissues,and this also indicates that the SBP family genes has certain regulatory functions during the development and flowering in citrus apex.