We have first developed an improved method to isolate nuclei from plant material, as the procedure to identify transcriptional regulators by DA-MS requires to isolate the proteins from nuclei of plant cells. This method allows to obtain intact nuclei containing proteins in their native conformation (it is to say transcription factors with DNA-binding capacity), and low presence of non-nuclear contaminants.
In summary, the procedure is based on the homogenization of the material, filtration of the homogenate, lysis of the filtrated using Triton X-100 to lyse non-nuclear organelles. The lysate is then subjected to a centrifuge on a discontinuous gradient based on a layer of 2.5 M sucrose and another layer of 60% Percale. The isolated nuclear fraction is then washed to obtain purified nuclei. The different steps of this methods have been optimized thanks to biochemical characterization of the different fractions, mainly by Western Blot analyses. The optimized method, that suits not only A. thaliana but also other plant species such as tomato, is described in a method paper that will be submitted soon.
In a second step, the nuclear proteins were isolated and used for DNA affinity. The factors bound to the target DNA probe (labeled F3 fragment) were digested and subjected to LC/MS/MS (Liquid Chromatography-tandem Mass Spectrometry). We used 1 g of dissected shoot apices where STM is normally expressed and also 1 g of developed leaves where STM is not expressed as negative control. We used three independent biological replicates. The analysis of the results from these experiments showed a lack of extensive contamination due to handling of samples or non-nuclear factors. From the nuclear factors obtained we found a group of factors that belong to the same clade of a family of plant transcription factors. As most of these factors have not been characterized yet, we started the functional characterization of them. We obtained insertion lines for the genes from seed stocks (ABRC – Arabidopsis Biological Resources Center) and we are determining through genotyping, sequencing and expression analyses the null alleles for these genes. Through phenotyping we are characterizing the role of these genes. Due to gene redundancy typical in plants, we might need to generate higher order mutants to reveal a phenotype. In parallel we are generating promoter reporter lines for these genes to study their expression patterns, and also generating lines that ectopically express the genes found in our study of factors binding the F3 region of STM promoter.
A research article characterizing the role of the plant factors found is expected once the functional assays will be done.
