Whole-genome Identification Of AP2,CBL,and CIPK Family And Functional Study Of CpCBL8 In Chimonanthus Praecox

The concentration of intracellular Ca²⁺will be changed when plants are subjected to environmental stress.CBL,a calcium-dependent sensing protein,is activated by a conformational change after sensing and binding Ca²⁺,and then binds and activates the downstream target protein CIPK,constituting a CBL-CIPK signaling system to generate a cascade response downstream in response to the stress.The AP2 family belongs to the AP2/EREBP transcription factor superfamily,and its members play an important role in plant growth and development and response to environmental stress.Wintersweet（C.praecox）is a traditional and valuable flowering tree in China,and also a rare winter-flowering garden plant with high ornamental and economic value.The identification and function analysis of AP2,CBL,and CIPK families is of great significance for genetic improvement and flowering regulation in C.praecox.In this study,the genome-wide identification of AP2,CBL,and CIPK family members was performed using a combination of local BLAST and local HMMER,and the identification results were analyzed using a variety of bioinformatics research tools.The expression data of AP2,CBL,and CIPK family members in the transcriptome database were screened in flower buds dormancy breaking and enlargement by prolonged cold exposure in C.praecox,and their expression patterns during flower development were analyzed.The Cp CBL8 and Cp CIPK9 with significant changes in expression patterns in the transcriptome database were focused on and their expression profiles were analyzed.Ca Cl₂ solution was sprayed on flower buds to explore the effect of exogenous Ca²⁺on the flowering stage and the changes of Cp CBL8 and Cp CIPK9 expression in C.praecox.The phenotypic and resistance characteristics of 35S::Cp CBL8/Arabidopsis lines were analyzed by ectopic overexpression of Cp CBL8 and the molecular mechanism of the corresponding changes in Arabidopsis was speculated.The main findings were as follows:（1）20 Cp AP2s,9 Cp CBLs,and 22 Cp CIPKs were identified.Gene structure analysis showed that the number of Cp AP2s introns ranged from 6 to 10 and Cp CBLs introns ranged from 7 to 11,while Cp CIPKs can be classified into few-and multiple-intron groups with 0～3 and 11～14 introns,respectively.The promoter sequence analysis showed that all three families have cis-acting elements in response to hormones and stress,and some Cp AP2s and Cp CIPKs are also involved in plant growth and development.Protein interaction prediction analysis showed that each Cp CBL has multiple target Cp CIPKs;Gene Ontology（GO）enrichment prediction analysis showed that Cp CBLs and Cp CIPKs are mainly involved in ion binding,signal transduction,and stress response.Synteny analysis showed that there are 17 Cp AP2s,6 Cp CBLs,and 6 Cp CIPKs forming26,7,and 4 pairs of duplication genes respectively,all of them are whole-genome duplication or segmental duplication and are subject to evolutionary purifying selection.（2）The analysis of transcriptome data showed that most Cp AP2 family members were highly expressed only in the perianth segment and pistil/stamen primordium formation stage（FB.Apr/May）,while low in the stage when flower buds（FBs）underwent chilling requirement（CR）accumulation.The expression of Cp AP2-11,Cp CBL2/5/6/8/9,and Cp CIPK2/5/6/13/16/20 was higher in FBs in November（FB.Nov）,but as FBs began to undergo CR accumulation until reached 570 chill units（CU）,their expression showed down-regulation in FBs of initial blooming with 570 CU（IB570）in some degree;the expression of Cp CBL1/7 and Cp CIPK3/8/9/10/11/14/17 increased during the process of CR accumulation comparing to that of in FB.Nov,then decreased in IB570,presumably,the above genes have negative regulation in the process of chilling-induced dormancy breaking,FBs enlargement,and blooming in C.praecox.The expression of Cp AP2-17,Cp CBL3/4,and Cp CIPK1/4/19/22 was elevated in IB570,and it was speculated that they might play a positive regulatory role in the FBs enlargement.Trend analysis of FB150-FB300-FB450-IB570 showed that the expression of Cp CBL8 and Cp CIPK9 decreased in IB570 compared to that in FB150/300/450,while Wenn analysis showed that their expression in IB570 was significantly different from that in samples of other periods.Polarized Zeeman flame atomic absorption spectrophotometer（FAAS）determination of Ca²⁺content in different samples revealed that the Ca²⁺content in FBs of initial blooming period（IB）under natural conditions or that of IB570 under artificial chilling exposure was significantly lower than other samples,under corresponding conditions,respectively;and the expression of Cp CBL8 and Cp CIPK9 are correlated with the Ca²⁺content.（3）After exogenous Ca²⁺（Ca Cl₂）treatment of FBs,it was found that the opening time was significantly delayed by 30 or 50 m M Ca²⁺treatment,while the expression of Cp CBL8 in FBs of all treatments and the control was significantly lower in the IB than that of in FBs rupture period.The expression of Cp CBL8 and Cp CIPK9 in 30 and 50 m M Ca²⁺treated FBs were significantly higher in IB compared to that in the control and 10and 20 m M Ca²⁺treatments,and it is speculated that Cp CBL8 and Cp CIPK9 may be involved in the negative regulation of flower development in C.praecox.（4）The expression profile revealed that the relative expression of Cp CBL8 was significantly higher in leaves,followed by buds,and the least in flowers;in different parts of flowers,which was significantly higher in the middle/inner tepals than that in the outer tepals,stamens,and pistils.While the expression of Cp CIPK9 was significantly higher in flowers and different parts of the flower than that in roots,stems,leaves,and fruits.12-leaf-old seedlings of wintersweet were treated by abiotic stress and exogenous hormone,and the expression of Cp CBL8 was found to be up-regulated under induction by low temperature,drought,salt,abscisic acid,or methyl jasmonate compared to control,except for that induced by high temperature;while Cp CIPK9 expression was elevated under high/low temperature,salt,drought,abscisic acid,methyl jasmonate,and salicylic acid induction.Subcellular localization assays showed that Cp CBL8 was localized at the plasma membrane and nucleus,and Cp CIPK9 at the plasma membrane,cytoplasm,and nucleus.Yeast two-hybrid assays showed that both Cp CBL8 and Cp CIPK9 did not possess the transcriptional self-activation activity and were able to specifically interact with each other.（5）The constructed 35S::Cp CBL8-2301G expression vector was transformed into Arabidopsis/Col-0 to obtain overexpression（OE）lines.Phenotypic observation of the screened homozygous T2 generation OE lines revealed that the flowering period was significantly delayed compared with WT（Col-0）,the rosette leaf numbers,leaf areas,and the inflorescence were significantly increased,and the higher the expression of Cp CBL8,the more significant the phenotypic changes.Determination of flower development-related endogenous genes revealed that the expression of SOC1,AP1,and LFY was significantly down-regulated in the OE lines compared to WT.（6）Sowing each line in MS medium simulating salt and drought stress revealed that the OE lines had higher seed germination rate and longer seedling roots compared to WT.Salt and drought stress assays showed that the OE lines wilted less,had higher superoxide dismutase activity,relative water content,chlorophyll,and proline content,as well as less stomatal conductance,malondialdehyde content and reactive oxygen species accumulation,and lower electrolyte leakage compared to WT,while the results of all physiological indicators measured under low-temperature stress were opposite completely.（7）Lower Fv/Fm andΦ_PSⅡwere detected in 35S::Cp CBL8/Arabidopsis OE lines compared to WT under low-temperature stress at-5℃for one hour.Determination of endogenous genes of the low-temperature response pathway revealed that the expression of CRPK1 and 14-3-3λwas significantly up-regulated,and the expressions of CBF1/2/3and COR15A/15B/47 were significantly down-regulated in the OE lines compared to WT.In conclusion,the genome encodes 20 Cp AP2s,9 Cp CBLs,and 22 Cp CIPKs in C.praecox.Cp CBL8 is a positive regulator of salt and drought stress response and is a potential gene to improve salt and drought tolerance,and the Cp CBL8-Cp CIPK9signaling system may be involved in the negative regulation of flower buds enlargement in C.praecox.Heterologous expression of Cp CBL8 in Arabidopsis enhances salt and drought tolerance in OE lines,but makes it more sensitive to low-temperature stress and has a suppressive effect on flower development.Cp CBL8 expression can be induced by spraying Ca Cl₂,which in turn can be used to regulate flowering in C.praecox.