Characterization of MYB homologous protein in tobacco pollen

Abstract

A mixture of oligonucleotides corresponding to sequence from the highly conserved MYB domain were used as probes to screen a cDNA library prepared from poly $A^{+}$ RNA isolated from mature tobacco pollen. Two clones were isolated and sequenced (Ntmybps1 and Ntmybps2) which revealed the presence of two MYB domain repeats characteristic of most plants. Detailed spatial and temporal northern blot analysis showed that the Ntmybps1 transcript was present specifically in mature and germinating pollen and was first detectable immediately before the asymmetrical division pollen mitosis I (PMI). Southern blot analysis revealed that Ntmybps1 and Ntmybps2 represent single copy genes and members of a small gene family in the tobacco genome. In planta functional analysis of NtMYBPS1 showed that the N-terminal MYB domain was sufficient to direct a MYB-GFP fusion protein to the nucleus. NtMYBPS1 expressed in E.coli was shown to recognise two MYB binding sequences and to activate the transcription of two phenylalanine ammonia lyase (PAL) promoters fused to the GUS reporter gene using tobacco leaf protoplasts. We therefore postulate that Ntmybps1 gene product represents a functional pollen-specific transcription factor.