The analysis of entire gene promoters by surface plasmon resonance.

We demonstrate that the biophysical technique of surface plasmon resonance (SPR) analysis, which has previously been used to measure transcription factor binding to short DNA molecules, can also be used to characterize interactions involving entire gene promoters. This discovery has two main implications that relate, respectively, to novel qualitative and quantitative uses of the SPR technique. Firstly, SPR analysis can be used qualitatively to test the capacity of any transcription factor to interact physically with its putative target genes. This application should prove particularly useful for the confirmation of predicted transcriptional interactions in model species, and for comparative studies of non-model species in which transcriptional interactions are not amenable to study by other methods. Secondly, SPR may be used quantitatively to characterize interactions between transcription factors and gene promoters containing multiple cis-acting sites. This application should prove useful for the detailed dissection of promoter function in known target genes. The qualitative and quantitative applications of the SPR analysis of whole promoters combine to make this a uniquely powerful technique, which should prove particularly useful in systems biology, evolutionary developmental biology and various branches of applied biology.

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins.

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron.

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.