Periodic Reporting for period 1 - MASAM (New tools for key questions in plant development: using qualitative and quantitative proteomics for direct determination of the set of DNA and RNA-binding factors regulating single copy genes)
Okres sprawozdawczy: 2015-11-01 do 2017-10-31
Plant development relies on the activity of meristems, a structure that contains stem cells that form plant organs. The shoot apical meristem (SAM) located at the growing tip generates all the aerial parts of plants through differentiation. To understand how the activity of the SAM is regulated it is then important to focus on the regulation of the activity of key genes acting in the SAM. SHOOT MERISTEMLESS (STM) from plant model Arabidopsis thaliana is known to be required for the maintenance of the activity of the shoot apical meristem. However the factors that bind regulatory regions of STM are largely unknown.
The overall objectives of the project are to determine the factors that regulate the expression of STM. For this purpose we used a proteomic approach, using DNA-binding affinity coupled with mass spectrometry (DA-MS). We focused on the regulatory region F3 from STM promoter that was stated as important for STM regulation previously by the experienced researcher. Another goal in this project is to establish this procedure to be used in other regulatory regions of genes to study biologically relevant processes.
The overall objectives of the project are to determine the factors that regulate the expression of STM. For this purpose we used a proteomic approach, using DNA-binding affinity coupled with mass spectrometry (DA-MS). We focused on the regulatory region F3 from STM promoter that was stated as important for STM regulation previously by the experienced researcher. Another goal in this project is to establish this procedure to be used in other regulatory regions of genes to study biologically relevant processes.
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.
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.
The determination of factors that bind a DNA sequence of interest through DA-MS has not been extensively used in plant biology. We have developed a procedure with the aim to extend this type of analysis in future studies. Many biological processes depend on gene activity and this activity is mainly regulated at the level of gene transcription. We have focused in the study of shoot apical meristem development regulated by STM through the activity of factors binding the F3 promoter fragment. Our goal is that this procedure can be used for the study of many other biological processes.