Functional conservation of male germline regulators in flowering plants

The production and differentiation of twin sperm cells in pollen grains of Arabidopsis thaliana, is critically dependent upon the R2R3 MYB transcription factor DUO1 POLLEN1 (DUO1) and one of its direct targets, DUO1-ACTIVATED ZINC FINGER 1 (DAZ1). Genes encoding DUO1 and DAZ1 orthologues are present throughout the angiosperms including tomato (Solanum lycopersicum). A major objective of the work presented was to validate the suggestion that tomato orthologues, SlDUO1A, SlDUO1B and SlDAZ1, possess the same male germline functions as their Arabidopsis counterparts in vivo. Transcript profiles for SlDUO1A, SlDUO1B and SlDAZ1 were similar to those established for their Arabidopsis orthologues in developing pollen. The analysis of tomato TILLING mutant lines indicate that SlDUO1A, SlDUO1B have redundant functions, while strong genetic evidence is presented to support an essential contribution of DAZ1 to male germline development. Furthermore, the conserved function of a strong transcriptional activation domain, which resembles the Nine amino acid Transactivation domain (9aaTAD) family, was demonstrated for SlDUO1A. The second main aspiration was to exploit the natural variation in 855 accessions of A. thaliana to investigate the in vivo role of conserved repression motifs in DAZ1. The function of a naturally truncated DAZ1Ler allele was studied by analysing the phenotype of CRISPR-Cas9 generated knockout lines and in complementation assays of daz1 daz2 mutants. When male germline development was under the control of DAZ1Ler, a distinct generative cell mitotic arrest phenotype was observed.

The data highlight a significant role for the ethylene-responsive element binding factorassociated amphiphilic repression (EAR) motif for mitotic progression of the generative cell. These findings provide new insight into the conservation of the DUO1-DAZ1 regulatory module in tomato and hidden details about the machinery that controls generative cell division and sperm cell differentiation that is required for double fertilisation, seed production and ultimately for food security.