The Role of DOF6 in Programmed Cell Death in the Arabidopsis Endosperm

Programmed cell death of the endosperm is a normal process in plants in which a transcription factor encoded by the ZHOUPI (ZOU) gene is required. In zou mutants the seed has an unusually persistent endosperm and the embryo is smaller than normal. The target genes that mediate this role of ZOU are not known. Transcriptional profiling of wild-type and zou mutant seeds identified DOF6 as a target gene. DOF6 is a member of a large, plant specific group of transcription factors, but mutants are not available. The aim of this project was to test the hypothesis that DOF6 mediates the function of ZOU in endosperm cell death by 1) testing whether expression of a DOF6 transgene in zou mutant seed can rescue the defective endosperm phenotype and 2) test whether inactivating DOF6can mimic the zou mutant phenotype. To address 1) we first identified a ZOU promoter fragment sufficient to drive expression of DOF6 cDNA in endosperm. We then introduced pZOU::DOF6 transgene constructs into zou mutants by floral dip transformation. To address 2) WMD3 programme was used to design artificial microRNAs (amiRNAs) targeting different regions of the DOF6 and ZOU itself (as a control producing the typical zou mutant phenotype when introduced to wild-type). Four specific oligonucleotides for each amiRNA were generated and then site-directed mutagenesis was employed to introduce the amiRNA sequences into the pRS300 precursor on a template plasmid. The amiRNA precursors were then cloned into a gateway entry plasmid and consequently into a binary plasmid under the control of the ZOU promoter. Arabidopsis (Col-0) plants were transformed with the resulting plasmid vectors using the Agrobacteriumfloral-dip method.The amiRNA constructs were successfully generated. Screening of the seeds in the next generation for kanamycin resistance proved the vectors were integrated and transformation was successful. Moreover, it was shown that a 5 kb ZOU promoter fragment can rescue zou mutant phenotype when driving ZOU cDNA and thus recapitulates the ZOU expression patterns. Therefore, it is sufficient to drive DOF6 cDNA and DOF6 amiRNA constructs. Further investigation is required to confirm phenotypes associated with these constructs. Findings from this study are an important first step towards the characterization of the DOF6 gene in Arabidopsis thaliana, but further investigation is required in order to fully understand its role. The identification of the genes that control endosperm cell death will be of practical importance as the bulk of human food is provided by endosperm of cereals.