Physiological Function And Breeding Application Evaluation Of Tea Plant CsAPL1 In Flower Formation

Camellia sinensis（L.）O.Kuntze belongs to the genus Camellia of the Theaceae,and it is also an important leaf economic crop in China.Generally,vegetative growth and reproductive growth run through the whole life cycle of tea plants,and the stage of reproductive growthbeginning with flower formation.However,vegetative growth and reproductive growth are in a competitive relationship.The long andvigorous flowering and fruiting of tea plants will consume a large amount of nutrients which affect the yield and quality seriously,and restrict the improvement of economic benefits of tea industry.Therefore,it is of great significance to elucidate the molecular regulatory mechanism of teaflower formation and breeding of new cultivars with few flowers or no flowers to promote the vegetative growth and finally improving thequality and yield.Based on the previous transcriptome and genomedatabase of tea plants,we searched and obtained a protein with MADS and K-box conserved domains,which is encoded by the Cs APL1 gene.In addition,the expression of Cs APL1 was upregulated in response to the development of floral organs,suggesting that it may be a key regulatory gene involved in flower formation of tea plants.In this study,based on the cloning and isolation of Cs APL1 gene from tea plants,we analyzed sequence characteristics,expression patterns,physiological functions,regulatory mechanisms,and application evaluation in tea cultivars with different flowering characteristics.The main findings are as follows:1.The Cs APL1 gene sequence was cloned and obtained by PCR amplification using the c DNA of tea plant‘Longjing 43’as a template,and the length of the coding sequence of the Cs APL1 gene was 693 bp,encoding 230 amino acid residues.Further analysis of protein structure characteristics showed that the molecular formula of Cs APL1 protein is C₁₁₄₉H₁₈₈₀N₃₄₆O₃₅₅S₈,the molecular weight is 26.5 k Da,the theoretical isoelectric point is 8.95,and Cs APL1 protein has conserved MADS and K-box domains.Phylogenetic tree analysis showed that Cs APL1 had high homology with Arabidopsis AT5G60910.1（AGL8/FUL）,AT1G26310.1（AGL10/CAL）,AT1G69120.1（AGL7/AP1）and AT3G30260.1（AGL79）,all belonging to AP1-like subfamily.2.The expression levels of Cs APL1 gene in different tissues andexogenous treatments of tea plants were detected.The results showed that the expression of Cs APL1 gene was the highest in mature leaves in spring.In autumn,the expression level of Cs APL1 gene was the highest inblossom buds,and the expression level significantly decreased inalabastrums and blooming flowers.The expression level of Cs APL1 gene in petals,stamens,pistils,calyxes and peduncles of flowering organs was low.The expression of Cs APL1 gene was significantly upregulated with the prolongation of 4°C low-temperature treatment time.Thetranscription level of Cs APL1 gene under long days was always higher than that under short days.The expression of Cs APL1 gene significantly increased after exogenous ABA and GA3 treatment for 6 hours.These results suggested that Cs APL1 gene functions mainly in the early stage of floral organ development and was involved in responding to lowtemperature,photoperiod and hormone signals.3.The Cs APL1 promoter sequence with 1416 bp length was cloned and obtained,and cis-acting element analysis showed that the Cs APL1promoter was rich in multiple light response,stress response andhormone response elements.GUS staining analysis of Pr Cs APL1::GUS transgenic Arabidopsis showed that GUS staining was evident only in leaves among all vegetative organs,and the GUS activity in matureleaves was higher than that in young leaves.Among all reproductiveorgans,GUS staining was found only in anthers of blossom buds,but not in alabastrums and blooming flowers.Long-day and exogenous GA3,ABA treatments induced more GUS expression in leaves.Our results further indicated that Cs APL1 gene plays an important role in early flower bud development.4.Subcellular localization analysis showed that Cs APL1transcription factor was localized in the nucleus.Overexpression of Cs APL1 in Arabidopsis showed early flowering phenotype under long-day and short-day conditions,and the germination rate of seeds decreased significantly.The results of q RT-PCR detection showed that the expression levels of At LFY,At SOC1,At FT,At ABI5,At FCA,At FY,At SPL3,At SEP3,At FLD and At VRN2 genes were significantly upregulated inCs APL1 overexpressing Arabidopsis.These results suggest that Cs APL1gene promotes flowering in Arabidopsis by regulating multipleflowering-related genes.5.Yeast two-hybrid system and firefly luciferase complementation imaging assay showed that Cs APL1 protein could interact with Cs SVP1,Cs SVP2,Cs SOC1-L,Cs AP1 and Cs AGL6 proteins to form proteincomplexes,respectively.Yeast one-hybrid system and dual-luciferase reporter system found that Cs COL transcription factor binds to the promoter of Cs APL1 gene and then induces its expression.The results indicated that Cs APL1 interacted with multiple flowering factors to synergistically regulate the flowering process of tea plants.6.Based on the field investigation of flowering traits,observation of axillary/floral bud tissues and expression changes of Cs APL1 gene infloriferous cultivars‘Zhonghuang 2’,‘Fudingdabaicha’and oliganthous cultivars‘Pingyangtezao’and‘Hongyafoshou’,the study found that the expression level of Cs APL1 gene in floral bud differentiation and floral organ development stages of the floriferous cultivars was significantly higher than that of the oliganthous cultivars,especially in cultivar‘Zhonghuang 2’with dense flowering and a long flowering period.The results showed that the expression level of Cs APL1 was positivelycorrelated with the number of flowering in tea plants,which could be applied to the early breeding and identification of tea cultivars with few（no）flowers.