We present a comprehensive overview on flavonoid-related phenotypes of A. thaliana tt and tds mutants, provide tools for their characterisation, increase the number of available alleles and demonstrate that tds3 is allelic to tt12 and tds5 to aha10. Flavonoid biosynthesis is one of the best-studied secondary metabolite pathways in plants. In the model system Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes: flavonol glycosides, anthocyanins and proanthocyanidins (PAs). PAs appear brown in their oxidised polymeric forms, and most A. thaliana mutants impaired in flavonoid accumulation were identified through screens for lack of this seed coat pigmentation. These mutants are referred to as transparent testa (tt) or tannin-deficient seed (tds). More than 20 mutants of these types have been published, probably representing most of the genes relevant for PA accumulation in A. thaliana. However, data about the genes involved in PA deposition or oxidation are still rather scarce. Also, for some of the known mutants it is unclear if they represent additional loci or if they are allelic to known genes. For the present study, we have performed a systematic phenotypic characterisation of almost all available tt and tds mutants and built a collection of mutants in the genetic background of the accession Columbia to minimise effects arising from ecotype variation. We have identified a novel tt6 allele from a forward genetic screen and demonstrated that tds3 is allelic to tt12 and tds5 to aha10.
This chapter describes a transient protoplast co-transfection method that can be used to quantitatively study in vivo the activity and function of promoters and promoter elements (reporters), and their induction or repression by transcription factors (effectors), stresses, hormones, or metabolites. A detailed protocol for carrying out transient co-transfection assays with Arabidopsis At7 protoplasts and calculating the promoter activity is provided.
Transcription initiation of the genes coding for small nuclear RNA (snRNA) has been extensively analyzed in human and fruit fly, but only a single ortholog of a snRNA-activating protein complex (SNAPc) subunit has so far been characterized in plants. The genome of the model plant Arabidopsis thaliana encodes orthologs of all three core SNAPc subunits, including AtSNAPc4 - a 4R-MYB-type protein with four-and-a-half adjacent MYB repeat units. We report the conserved role of AtSNAPc4 as subunit of a protein complex involved in snRNA gene transcription and present genetic evidence that AtSNAPc4 is an essential gene in gametophyte and zygote development. We present experimental evidence that the three A. thaliana SNAPc subunits assemble into a SNAP complex and demonstrate the binding of AtSNAPc4 to snRNA promoters. In addition, co-localization studies show a link between AtSNAPc4 accumulation and Cajal bodies (CB), known to aggregate at snRNA gene loci in humans. Moreover, we show the strong evolutionary conservation of single-copy 4R-MYB/SNAPc4 genes in a broad range of eukaryotes and present additional shared protein features besides the MYB domain, suggesting a conservation of the snRNA transcription initiation machinery along the course of the eukaryotic evolution.
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