Mechanism Of LoMYB21 And LoSPATULA Genes Regulating Anther Dehiscence In Lily

Lily has high ornamental and economic value.Pollen contamination is a serious problem to be solved in lily,and it is closely related to anther dehiscence.Many factors affect anther dehiscence,such as plant hormones,temperature,water,and so on.However,the mechanism anther dehiscence is largely focused on endothecium lignification but seldom on stomium degeneration.Lily anther dehiscence is seldom investigated and largely unknown.In this study,we cloned an anther dehiscence-related gene Lo MYB21 from lily cultivar ’Siberia’ based on transcriptome and bioinformatics analysis,and analyzed its function in regulating anther dehiscence.Through further research,the interaction protein Lo SPATULA of Lo MYB21 was discovered,and the function of Lo SPATULA was studied.The results will provide a scientific basis for the breeding of Lily without pollen contamination.The main results and conclusions are as follows:(1)Morphological characteristics of the stomium zone from different developmental stages of ‘Siberia’ lily anthers were investigated,stomium zone lysis occurred when flower buds were 6–8 cm in length and was completed in 9 cm.Transcriptome sequencing was performed on 5,6,7,8,9 cm stomium tissues,and metabolomics detection was performed on 6,7,8 cm stomium tissues.Transcriptomics and metabolomics analysis identified genes involved in stomium zone degeneration,including those associated with hormone signal transduction,cell structure,reactive oxygen species(ROS),and transcription factors.In addition,TUNEL(Td T-mediated d UTP nick end labeling)assays showed that programmed cell death was important during anther stomium zone degeneration.(2)JA and Me JA content was the highest at the critical stage of lily anther dehiscence.In addition,Me JA treatment promoted anther dehiscence process,whereas JA biosynthesis inhibitor sodium diethyldithiocarbamate(DIECA)inhibited anther dehiscence.Through transcriptome sequencing,we analyzed the expression pattern of MYB transcription factors in stomium zone during anther dehiscence.Then,Lo MYB21,a R2R3 MYB transcription factor related to JA signal transduction,was isolated and identified in lily.Lo MYB21 is homologous to At MYB21 of Arabidopsis thaliana.It was localized in the nuleus and cytoplasm and showed transcriptional activation at C terminal in yeast cells.Furthermore,Lo MYB21 was highly expressed at late anther development stage;situ hybridization results showed that it had fluorescence signals in the region of stomium during anther dehiscence.With silencing of Lo MYB21 in lily,normal anther dehiscence process was delayed,which caused reduction in JAs accumulation.With overexpression of Lo MYB21 in Arabidopsis,anther dehiscence process also was delayed,and JA biosynthesis pathway genes expression decreased.These results suggest that Lo MYB21 act as a switch involved in JA synthesis to control anther dehiscence.(3)To further explore the regulatory network of Lo MYB21 in anther dehiscence,Lo SPATULA was screened according to the expression pattern of transcriptome transcription factors.Yeast transformation experiment,firefly luciferase complementation image assay and bimolecular fluorescence complementation experiment found that Lo MYB21 interacted with Lo SPATULA protein.Lo SPATULA is a transcription factor of b HLH family,and has the closest relationship with SPATULA of Elaeis guineensis and Phoenix dactylifera,and N-teminal contained a acidic domain.Transcription factor Lo SPATULA subcellular localization is located on the nucleus,transcriptional activation activity occured in yeast cells.In addition,the gene expressed in lily anther stomium zone.Lo SPATULA overexpression promotes anther dehiscence process and JA biosynthesis in Arabidopsis.