Cytokinin Response Regulators Regulate The Root Morphogenesis In Citrus

Citrus is the most important fruit in China with the largest planting area and production.Citrus orchards are mainly distributed in mountainous and hilly areas,and the growth of citrus is vulnerable to drought,salinity and other adverse conditions.Root is the first organ of the plant to sense the changes of soil environmental conditions,which plays an important role on the response to abiotic stress.Citrus is mainly propagated by grafting.There are significant differences in root morphological structure among the various citrus rootstocks.The morphological structure of the root not only has a decisive influence on the ability of root to absorb water and nutrients,but also has an important influence on the resistance of root system.Therefore,it is of great significance to study the root morphogenesis of citrus and explore the regulation mechanism of formation of root system architecture for the sustainable development of citrus industry.In the previous study,transcriptome sequencing analysis of citrus rootstocks with different root lengths revealed that cytokinin response regulators(RRs)may be involved in root system architecture.However,in woody plants,RR members have been identified in poplar,apple,pear,peach etc.,but there are only few reports on study of their functions.However,such studies have not been reported in citrus.In view of the importance of root morphogenesis in citrus,identification of the function of RR gene in citrus has been conducted in this study.Of 29 RR genes were identified from citrus by genome-wide identification of RR family members and termed as Cc RRs.Cc RRs that may be involved in root growth and development were screened by bioinformatics and expression analysis.The genetic transformation of citrus was carried out to verify the function of candidate Cc RRs in regulating the growth and development of citrus roots.The regulatory relationship between two candidate factors(Cc RR5 and Cc RR14)was revealed by analysis of promoter sequence and transient transformation of tobaccos.The key component Sucrose Non-fermenting Protein Kinase 2(Sn RK2s)of ABA signaling pathway were identified to interact with Cc RRs by yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC)experiments.In addition,the regulation of Sn RK2 s member Cc Sn RK2.10 on citrus root growth and development was also found.The main results of this study are as follows:1.A total of 29 RR family members were identified in citrus.A total of 29 RR family members were identified from the clementina genome(Citrus clementina),including 21 RR proteins and 8 pseudo RR proteins,which were named Cc RR1-21 and Cc PRR1-8,respectively,according to their positions on the scaffolds.Phylogenetic analysis was performed with RRs from Arabidopsis(24 ARR and 9 APRR),poplar(22 Pt RR and 11 Pt PRR)and citrus(21 Cc RRs and 8 Cc PRRs).According to the amino acid sequence and characteristics of the domains,RRs in citrus can be classified into four types.Type A Cc RRs have seven members,namely Cc RR5-6,Cc RR8,Cc RR11-13 and Cc RR19,all of which have a D-D-K domain.C-type Cc RRs have four members(Cc RR3,Cc RR7 and Cc RR17-18)with a D-D-K domain similar to that of in type A.Type B Cc RRs have the most members(Cc RR1-2,Cc RR4,Cc RR9-10,Cc RR14-16 and Cc RR20-21),and all of them contain a MYB-like DNA binding domain except the conserved D-D-K structure.Cc PRRs are distributed on both sides of B-type Cc RRs,with a total of 8 members,all of which have a D-D-K domain and most of them have a CCT domain.The cis-acting element analysis of the promoter showed that the promoter of Cc RRs contained a large number of light response elements,multiple hormone response elements,drought induction,low temperature response,defense and stress response elements,as well as meristem development and endosperm expression elements,indicating that they may be involved in the regulation of citrus growth and development,and abiotic stress resistances.Among members of the type A and type B Cc RRs,Cc RR4,Cc RR5,Cc RR6 and Cc RR16 were highly expressed in roots and leaves,and have obvious responses to hormone and abiotic stress treatments.Cc RR2,Cc RR10,Cc RR14 and Cc RR19 were highly expressed in roots in response to hormones and stress treatments.There were significant differences in the expression of the above eight Cc RRs in three zones of the root,indicating that their functions in regulating root growth and development were different.The nine citrus rootstocks(Poncirus trifoliata Pt026,Pt030,Pt034,Pt038,C.wilsonii Tanaka ’Zhique’,C.limonia Pasquale ’Volkamer’,C.limonia Osbeck ’Canton Lemon’,C.reshni Hortrt.Ex Tan ’Cleopetra’,C.reticulata ’Zhuju’)with different root system architectures were used to further analyze the three highly and differentially expressed genes.The results showed that the expression of Cc RR5,Cc RR10 and Cc RR14 was significantly correlated with the length of primary root,the number of lateral roots,the length of primary root and number of lateral roots,respectively.The results of this study showed that Cc RRs were involved in the regulation of citrus root growth and development,but the functions of each member were different.2.Cc RR5 and Cc RR14 were involved in the regulation of root morphogenesis.Subcellular localization analysis showed that Cc RR5 was mainly localized in the nucleus,and the signal on the cell membrane was weak,while Cc RR14 was only localized in the nucleus.Transcriptional self-activation experiments showed that Cc RR5 had no self-activation activity,while Cc RR14 had strong self-activation activity,indicating that Cc RR14 is a transcription factor.The 1500 bp-2000 bp promoter sequences of Cc RR5 and Cc RR14 were cloned.Cis-acting element analysis of Cc RR5 and Cc RR14 promoters showed that both of them contained stress response elements and MYBHv1 binding sites.In addition,the promoter of Cc RR5 also contains methyl jasmonate response elements,low temperature response elements,palisade mesophyll cell differentiation and endosperm expression elements,while the promoter of Cc RR14 contains zein metabolism regulation,auxin and ABA responsive elements and protein binding sites.The expression vectors of Cc RR5 and Cc RR14 promoters fused with GUS were constructed and genetically transformed into citrus to generate transgenic lines.GUS staining of transgenic citrus plants after rooting showed that both Cc RR5 and Cc RR14 can be expressed in roots and leaves of citrus plants.The overexpression and RNAi vectors of Cc RR5 and Cc RR14 were constructed and transformed into citrus.Through PCR verification and expression analysis,overexpression lines and interference lines of Cc RR5 and Cc RR14 were selected for rooting.Self-rooted plants were produced from three Cc RR5 overexpression lines,two interference lines,three Cc RR14 overexpression lines and two interference lines.The root phenotype of citrus transgenic lines and the expression level of the target gene were analyzed.The results showed that the transcription level of Cc RR5 was related to root length and lateral root number,and the expression of Cc RR14 also affected the root growth and development.3.Cc RR14 regulates the expression of Cc RR5,both them interact with Cc Sn RK2 s.The Cc RR5 promoter contains multiple Cc RR14 binding sites.Transient expression experiments in tobacco also showed that Cc RR14 can bind to the promoter of Cc RR5 and enhance its activity,indicating that Cc RR14 can regulate the expression of Cc RR5.The expression of Cc RR5 in Cc RR14 transgenic lines was significantly positively correlated with the expression of Cc RR14.Through genome-wide identification,seven Sn RK2 family members were found in citrus.The N-terminal kinase domains of all members were very conservative,and the C-terminal abiotic stress response and ABA induction-related domains were quite different.According to the amino acid sequence and domain characteristics,7Cc Sn RK2 s in citrus and 10 At Sn RK2 s in A.thaliana can be categorized into three groups,and named according to the homologous relationship with Sn RK2 s in A.thaliana(group 1: Cc Sn RK2.9,Cc Sn RK2.10;group 2: Cc Sn RK2.7,Cc Sn RK2.8,Cc Sn RK2a;group 3: Cc Sn RK2.3,Cc Sn RK2.6).The results of yeast two-hybrid and bimolecular fluorescence complementation experiments showed that Cc Sn RK2 s could interact with Cc RR14 and Cc RR5.Cc Sn RK2.10 can also interact with Cc RR14.4.Cc Sn RK2.10 can regulate root growth and development of citrus.Multiple transgenic lines were obtained by constructing overexpression and RNAi vectors of Cc Sn RK2.10 and genetic transformation of citrus.The expression of Cc Sn RK2.10 in roots of self-rooted transgenic citrus plants was measured.It was found that the expression of Cc Sn RK2.10 in the roots of overexpression plants was not significantly up-regulated,the overexpression effect was not significant.However,the interference effect in RNAi plants was extremely significant.The functional verification results of transgenic lines showed that silence of Cc Sn RK2.10 did not affect the rooting ability of the transgenic plants,but the new roots grew slowly,and the root length and root tip numbers were significantly lower than those of wild type plants,indicating that Cc Sn RK2.10 might positively regulate the growth and development of roots.5.Cc RR5,Cc RR14 and Cc Sn RK2.10 affected the expression of genes related to root growth and development.According to the studies reported in A.thaliana,the homologous genes related to root growth and development in citrus(Cc SHY2,Cc IAA17,Cc PIN3,Cc PIN7,Cc MYB44,Cc MYB77)were identified.The expression levels of these genes in transgenic lines were analyzed.It was found that the expression of Cc MYB77,Cc IAA17,Cc PIN3,Cc PIN7 was significantly correlated with the expression of Cc RR5.The expression of Cc MYB44,Cc IAA17,Cc PIN3,Cc PIN7 was also significantly correlated with the expression of Cc RR14.In addition,the expression of Cc RR5,Cc SHY2,Cc IAA17,Cc PIN3,Cc PIN7 and Cc MYB77 in transgenic lines was significantly correlated with the expression of Cc Sn RK2.10.The results indicated that the expression of Cc RR5,Cc RR14 and Cc Sn RK2.10 affected the expression of the genes related to root growth and development.The above results showed that Cc RR5,Cc RR14 and Cc Sn RK2.10 were involved in the regulation of root growth and development.In summary,the results of this study indicate that Cc RR5 and Cc Sn RK2.10 are positive regulators of root growth in citrus,and Cc RR14 also regulates root growth and development.Cc RR14 regulates the expression of Cc RR5,and both them interact with Cc Sn RK2 s to regulate the growth and development of roots to affect the architecture of the root system.