Functional conservation of a male germline regulatory module in crop plants

The formation of two sperm cells by the angiosperm male gametophyte is essential for seed production and ultimately for plant productivity. The MYB transcription factor DUO1 POLLEN 1 (DUO1) is a key regulator of male gametogenesis in Arabidopsis thaliana and forms a germline-specific regulatory module with the functionally redundant genes DUO1-ACTIVATED ZINC FINGER1 (DAZ1) and DAZ2. This regulatory module is conserved in some bryophytes, but its wider role among angiosperms remains unexplored. Here the DUO1-DAZ1 regulatory module is investigated in tomato (Solanum lycopersicum L.), wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). Two DUO1 orthologues and a single DAZ1 orthologue were identified in the genomes of tomato and barley, while three DUO1 and three DAZ1 homoeologues are present in the bread wheat genome. Analysis of RNA-seq data revealed that transcripts for DUO1 and DAZ1 are expressed in male reproductive tissues in each of these crops. Plants carrying knockout alleles disrupting SlDUO1 or SlDAZ1 function in tomato were shown to block generative cell division and the transmission of SlDUO1 or SlDAZ1 mutant alleles through pollen. Further, the generative cell was round in mutant alleles compared to elongated generative cell in wild type. Quantitative analysis of RNA expression in loss of function mutants also indicated that regulatory interactions of the DUO1-DAZ1 module are conserved in tomato. Preliminary analysis of knockout alleles disrupting TaDUO1 function in wheat indicate that generative cell division and sperm cell formation are affected. Plants homozygous for knock out alleles in one of two barley DUO1 orthologues were fertile and showed no generative cell defects. In summary, the data presented demonstrate an essential role for the DUO1-DAZ1 module in tomato and support the hypothesis that this regulatory module is more widely conserved in some key crop plants.