Integrative and self-replicating Lc vectors and their transactivation capacity in maize callus protoplasts

Abstract

The regulatory Lc gene is a member of the R gene family of maize which amongst other transcriptional activators controls anthocyanin biosynthetic genes. The availability of R locus mutants which lack anthocyanin production in all tissues offers the possibility of studying cell lineage by introducing a chimeric Lc gene into defined cells and following cell autonomous anthocyanin production. For this purpose integrative and self-replicating Lc vectors were constructed. Integrative expression vectors contained the 2.4 kbp Lc cDNA with the entire 5′ leader fused to the constitutive cauliflower mosaic virus 35S promoter with and without the maize alcohol dehydrogenase 1 intron 1 or to the mesophyll specific phosphoenolpyruvate carboxylase promoter of maize. To enhance expression and to circumvent the necessity of stable integration, extrachromosomally replicating and expressing wheat dwarf virus-Lc constructions were also designed. Both categories of expression vectors were tested in embryogenic callus-derived protoplasts of maize and were found to transactivate anthocyanin biosynthesis. Southern blot analysis indicated that the wheat dwarf virus-Lc constructions were replicating.