Abstract
In flowering plants the development of the male germ-line in pollen to form two functional sperm cells is essential for reproduction. DUO1 an R2R3 MYB transcription factor plays a key role in regulating genes required for germ-line division and specification into mature sperm cells. Mutant duo1 pollen contain a singular germ-like cell that cannot fertilize. Present studies of DUO1 have focused on the model organism Arabidopsis thaliana with regulation of DUO1 being of interest. This study tests if regulatory mechanisms are evolutionarily conserved across diverse flowering plant species with a focus on the legume Medicago truncatula.
A DUO1 orthologue in M. truncatula was able to complement the A. thaliana duo1 mutant through restoring mitotic function at pollen mitosis II. Specification and differentiation of the male germ-line was also restored as shown by successful male transmission from the complemented male germ-line. This indicated functional conservation between the two proteins.
In the A. thaliana DUO1 promoter a cis-regulatory module, Regulatory region of DUO1 (ROD1) is known to provide germ-line specific expression. Here analysis of the full length M. truncatula DUO1 promoter using a fluorescent promoter reporter showed male germ-line specific GFP fluorescence in A. thaliana during pollen development similar to that of the A. thaliana DUO1 promoter. Analysis of a 5ˊ deletion series of the M. truncatula DUO1 promoter then identified key regions necessary for expression with no GFP seen in a construct lacking the ROD1 module. The ROD1 regulatory module from M. truncatula was then isolated and used in a promoter reporter to show that M. truncatula ROD1 provides germ-line specific expression similar to A. thaliana ROD1. Collectively these results demonstrated that ROD1 is an evolutionarily conserved cis-regulatory module that determines germ-line specific expression of DUO1 critical for pollen development in dicotyledonous plants.
A. thaliana and M. truncatula DUO1 is thought to be able to auto-activate expression through DUO1 binding sites in the DUO1 promoter. This was explored through dual luciferase assays, however technical difficulties lead to inconclusive results and no clear conclusions could be made.