Functions And Molecular Mechanisms Of MdGRF11 In Regulation Of Apple Flower Bud Differentiation

Fuji apple is the first major apple variety in China.However,there are common problems in production,such as difficulties in forming flower buds on young trees and biennial bearing on mature trees,which seriously restrict the production efficiency and effectiveness of apple industry in China.It is important to excavate and deconstruct the key genes and their regulatory mechanisms in the process of apple flower bud differentiation for the genetic improvement and regulatory technology development of apple flower bud differentiation.In previous research,we screened for Md GRF11 protein that interacts with the flower-forming protein Md TFL1,which suggests growth regulatory factors（GRF,Growth-Regulating Factor）may be involved in the regulation of apple bud differentiation,but the related mechanisms need to be futher deconstruct.In current study,we used‘Nagafu No.2’as the test material to identify and characterize the expression of GRF gene family memebers.Meanwhile,we used heterologous genetic transformation of Arabidopsis thaliana to analysis their biological functions in regulating flower bud differentiation and analyzed the reciprocal proteins by means of yeast two-hybrid,firefly luciferase complementation imaging assay and other experimental techniques in oder to deconstruct the function and mechanism of Md GRF11 in regulating apple flower bud differentiation.The main results are as follows:（1）Genome-wide identification of GRF gene family members in apple.A total of sixteen Md GRF genes were identified in the Golden Delicious apple genome.These genes were located at eight chromosomes in apple,where chromosomes 2 and 15contained the most GRF genes.Multiple sequence alignment showed that Md GRF proteins contain two highly conserved QLQ and WRC domains in the N-terminal region,but C-terminus are highly variable in regard to the composition and number of amino acid residues.The phylogenetic analysis indicated that GRFs could be classified into six classes,and Md GRFs were distributed in five of these subfamilies（families II,III,IV,V and VI）.The results of promoter cis-acting analysis revealed that they have acting elements which are in response to gibberellin,auxin,methyl jasmonate,low temperature and light signal,while gibberellin,low temperature and light signal can play important roles in regulating flower bud formation,which implies that Md GRFs genes may participate in regulating apple flower bud differentiation.（2）Expression characterization and functional analysis of the apple GRF gene family.In the tissue-specific expression analysis of sixteen Md GRF genes,Md GRF1/4/6/9/10/11/14/16 genes showed a highly expression in apical buds on brachyblast during the period of physiological differentiation,whereas they were expressed at low levels in roots,stems,leaves,and fruits.Additionally,fruit load and GA₃ treatments inhibited the genes displayed various expression patterns at different time points during the period of physiological differentiation,in which Md GRF2/16and Md GRF11/12 were up-regulated and down-regulated by high fruit load respectively,while the transcript levels of Md GRF13/16 and Md GRF10/11/12 were higher and lower respectively in response to GA₃ treatment.Further,the function of Md GRF11 was analyzed.The subcellular localization results showed that Md GRF11was localized in the nucleus.We constructed a 35S:Md GRF11 overexpression vector and transformed into Arabidopsis（Col-0）,showed that Md GRF11 could promote early flowering,root elongation and leaf enlargement in Arabidopsis.（3）Molecular mechanisms of Md GRF11 in regulation of flowering in plants.Yeast two-hybrid and dual-luciferase assays confirmed that Md GRF11 can directly bind to the promoter of Md KNOX19 gene and inhibit its transcriptional activity.Yeast two-hybrid and luciferase complementation experiments demonstrated that the physical interactions between Md GRF9/11 and Md FT1/Md TFL1,and Md TFL1protein could interfere with the transcriptional repression of Md KNOX19 by Md GRF11.In summary,this study provides systematic data on the gene membership as well as expression patterns of the apple GRF family,and initially reveals that apple Md GRF11 is involved in regulating plant flowering through a pathway that co-regulates Md KNOX19 transcription by interacting with Md TFL1.These results enrich the molecular mechanism of apple flowering and provide a theoretical basis for improving apple horticultural traits through molecular breeding.