Functional study of transcription factor MYB305 in tobacco flower development

We isolated and characterized the cDNA encoding the ornamental tobacco (N.langsdorffii X N.sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This cDNA encodes a MYB family transcription factor protein containing a conserved R2R3 MYB DNA binding domain. This myb305 gene is expressed uniquely in floral organs with the highest level in the mature nectary and with lower levels in the ovary, floral tube, petals and flower abscission zone. A GFP-MYB305 fusion protein localizes to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrates that it functions as a transcription activator. A conserved 23 amino acid C-terminal domain is required to activate gene expression. Functional study discovered that MYB305 is involved in many physiological processes in plants.;First, MYB305 regulates expression of the major nectarin genes Nectarin I (nec1) and Nectarin V (nec5) in the nectary of ornamental tobacco plants. Temporally, myb305 gene expression precedes that of nec1 and nec5 genes. The purified GST-MYB305 proteins bind two consensus MYB-binding sites on the ornamental tobacco nec1 promoter as well as bind the single site located on the nec5 promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduce expression in nectary tissues. Ectopic expression of MYB305 in foliage is able to induce the expression of both nec1 and nec5. Further knockdown of MYB305 result in reduced expression of nec1 and nec5.;Second, MYB305 plays important roles in floral organ development and maturation by disrupting starch metabolism. In the MYB305 knockdown plants, the nectaries retain juvenile character and secrete reduced levels of nectar, the petals are smaller and do not fully expanded during anthesis. Abnormal starch metabolism has been found to contribute to these phenotypes because reduced starch accumulation was found in both the nectary, before nectar secretion, and in petals, before anthesis. The reduced levels of starch accumulation correlate with the expression of starch metabolic genes in both nectary and petal of MYB305 RNAi plants, suggesting that MYB305 is involved in starch metabolism by controlling the expression of starch metabolic genes.