The Casein Kinase 2β Subunit,CK2B1,is Required For Swollen Stem Formation In Brassica Juncea Var.Tumida

Brassica juncea var.tumida is an important stem-type variety of mustard in the Brassicaceae family.the formation of swollen stem is the determinant factor of the yield.the 8～15℃low temperature accumulation is the key environmental factor for swollen stem formation.However,the genetic mechanism and its regulation underlying swollen stem formation remains unknown,and the key genes controlling the swollen stem formation have not yet been mapped.in this study,we identified a casein kinase II beta subunit 1（CK2B1）and revealed its role in the swollen stem formation.Using Genome-Wide Association Analysis,gene function verification,protein interaction analysis and other methods.We found that CK2B1 regulates the swollen stem formation via cell number.The main results of this study are as follows:1)the CK2B1 was mapped based on Genome-Wide Association Analysis.The gene CK2B1 encodes the subunit of Casein Kinase 2.The RT-PCR assay of CK2B1 in the swollen stem/non swollen stem showed that CK2B1 expression level was significantly associated with the swollen stem formation.in addition,the expression level of CK2B1 in varieties with swollen stem was significantly higher than varieties without swollen stem.therefore,we hypothesized that CK2B1 was involved in the swollen stem formation.2)CK2B1 promoter variation and activity affect the swollen stem formation.Genotyping analysis showed that the variation of CK2B1 promoter was related to the swollen stem formation.We identified two types of CK2B1 promoter genotypes in swollen stem/non swollen stem,in which CK2B1 promoter was homozygous in swollen stem varieties and heterozygous in non-swollen stem varieties.the dual-luciferase reporter assay showed that the activities of CK2B1 promoter in swollen stem variety was significantly higher than that non-swollen stem variety,and was induced by low temperature.Meanwhile,Subcellular localization indicated that CK2B1 was localized in the nucleus,and GUS-staining revealed that CK2B1expression is visible in tissues such as seedling stage,leaf veins and stem nodes of rosette leaves.3)CK2B1 regulates swollen stem formation by affecting the cell number of pith.in CK2B1 silenced plant,the swollen stem formation was inhibited and correlated with CK2B1 expression level.A backcross population BC₄F₁crossed from a swollen stem line（STZ）and a non-swollen stem line（VC029）was constructed with STZ as the recurrent parent performed swollen stem phenotype.We observed the expression level of CK2B1 and swollen stem were significantly increased.the paraffin section results showed that the increasing of pith cells number was the main reason for swollen stem formation.in CK2B1 silenced lines,the cells number was significantly reduced.Accordingly,we indicated that CK2B1 regulates swollen stem formation via cell number control.4)CK2B1 regulated cell cycle control via phosphorylation of E2Fa.Yeast two hybrid and bimolecular fluorescence complementation assays showed that CK2B1 interacts with transcription factor E2Fa in the cell cycle regulation.Phosphorylation analyses in vitro showed that CK2 holoenzyme can phosphorylate E2Fa at sites of Tyr54,Tyr464,Thr63 and Ser431.these results suggested that CK2B1 regulated cell cycle via phosphoryaltion of E2Fa by CK2.Taken together,we mapped a CK2B1 that regulated swollen stem formation,and found that CK2 phosphorylated cell cycle associated transcription factors E2Fa to regulate the swollen stem formation.Consequently,we proposed a working model in which CK2 phosphorylates E2Fa to regulate swollen stem formation via the control of cell cycle.these findings help to elucidate the signals that control swollen stem and provide a promising molecular target to enhance yield of vegetative organ formation.