The strikingflower-in-flowerphenotype ofArabidopsis thalianaNossen (No-0) is caused by a novelLEAFYallele

Abstract

The transition to reproduction is a crucial step in the life cycle of any organism. In Arabidopsis thaliana the establishment of reproductive growth can be divided into two phases: In the first phase, cauline leaves with axillary meristems are formed and internode elongation begins. In the second phase, lateral meristems develop into flowers with defined organs. Floral shoots are usually determinate and suppress the development of lateral shoots. Here, we describe a Ds transposon insertion mutant in the Nossen (No-0) accession with severe defects in floral development and flower morphology. The most striking aspect is the outgrowth of stems from the axillary bracts of the primary flower carrying terminal secondary flowers. Therefore, we named this mutant flower-in-flower (fif). However, the insertion of the transposon in the annotated gene is not responsible for the fif phenotype. By means of classical and genome sequencing-based mapping, the mutation responsible for the fif phenotype was found to be in the LEAFY (LFY) gene. The mutation, a G-to-A exchange in the second exon of LFY, creates a novel lfy allele and causes a cysteine-to-tyrosine exchange in the α1-helix of the LFY DNA-binding domain.Whereas subcellular localization and homomerization are not affected, the DNAbinding of LFY FIF is abolished. We propose that the amino acid exchange interferes with the cooperative binding of LFY to its target DNA. To generate the strong fif phenotype, LFY FIF may act dominant-negatively by either forming non-binding LFY/LFY FIF heteromers or by titrating out the interaction partners, required for LFY function as transcription factor.

Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

“…The D fluorophore was mTRQ2, the A1 fluorophore was mVEN and the A2 fluorophore was mRFP. The vector for the expression of the A2-fusion proteins, was pB7RWG2-Dest by (Karimi et al, 2002), as it also contained the 35S promoter and was successfully applied in previous FRET-FLIM approaches (Boutant et al, 2010;Schoberer et al, 2013;Ladwig et al, 2015;Holzwart et al, 2018;Mohrholz et al, 2019). To facilitate the nomenclature, whenever protein X was labelled with D, protein Y was labelled with A1 and protein Z with A2, then only the sequence of the proteins will be written without mentioning the fluorophores: X-Y-Z.…”

“…For transformations with multiple constructs, an OD600 of 0.1 was set and mixed 1:1:1 with silencing inhibitor p19. Plants were watered and left to ambient conditions (24°C) with lid on top and imaged two days past transformation at the SP8 confocal laser scanning microscope (CLSM) (Leica Microsystems GMBH) with LAS AF and SymPhoTime software using a 63x/1.20 water immersion objective (Ladwig et al, 2015;Mohrholz et al, 2019). Data were derived from measurements of the lower epidermis, avoiding guard cells and stomata, with at least two biological replicates, comprising in average 20 data points and 11 data points for mTRQ2 -mRFP controls.…”