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AcronymBioregulators
  
TitleIdentification of molecular markers for the detection of bio-regulators that enhance plant productivity and quality
  
Duration1 August 2007 - 1 August 2010
  
Project leader Peter Eckes, Bayer CropScience GmbH, Germany [Company]
  

Project partners

John Graham Waples, BIOTEK Agriculture, France [Company]
Laurent Guerreiro, ARVALIS - Institute du végétal, France [Company]
Stefan Schillberg, Frauenhofer Institute for Molecular Biology and Applied Ecology, Germany
Paul Christou, Lleida University, Spain
José Luis Guardiola, Technical University of Valencia, Spain
   
FundingFrance National Institute for Agricultural Research (INRA)
 Germany  Research Centre Juelich – Project Management Juelich (FZJ-PTO) on behalf of the Federal Ministry of Education and Research (BMBF)
 Spain Ministry for Education and Science (MEC) 
  
  Total granted budget€ 2,179,822
  
Abstract 

We have identified a number of biologically active chemicals that influence plant growth and development by activating or inhibiting metabolism. The objective of this project is to exploit the potential of these bioregulators by identifying genes, proteins and metabolites that are up or down regulated when the chemicals are applied under stress or non-stress conditions and positively influence plant growth and productivity. Since comprehensive genomic resources are available for Arabidopsis thaliana and rice, gene expression in response to ten different active molecules will be analyzed using the Arabidopsis whole genome microarray and equivalent rice resources. Similar analysis of proteome and metabolome profiles will be carried out. Profiles will be compared to the biochemical and morphological effects of bioregulator application, resulting in the identification of genes, proteins and metabolites that indicate improved plant growth. These studies will include cell-based in vitro assays and greenhouse tests.
The suitability of these markers will be verified in important crop species. Orthologs will be identified in maize, wheat, rapeseed and vegetables using molecular biology techniques and in silico analysis. The crops will then be treated with the appropriate chemicals and assessed for expression profiles as well as biochemical, physiological and morphological characteristics as described above. In addition, chemicals will also be tested under field conditions using maize, wheat, rapeseed and vegetables.
Universal markers identified in these experiments will be used to establish a cell-based high-throughput assay. Promoters driving the marker genes will be fused to a fluorescent protein gene and plant expression cassettes will be introduced into Arabidopsis and rice plant suspension cells. The cell-based fluorescence assay will be verified using the already identified bioregulators and will allow the identification of novel or superior compounds that enhance crop yield and quality, which will be of significant benefit for the crop production markets. Moreover, markers for improved plant growth could also be used to select new plant lines with sustainable yield stability under biotic and abiotic stress. The successful implementation of this project will reflect the increasing impact of plant genomics on applied plant biotechnology.

  
Progress
Stress assays for different plant species have been established. The growth of Arabidopsis plants both on soil and in hydroponic cultures under stress-free conditions has been established and monitored. Stress assays for cold (on soil), salt and drought stress (hydroponic) are established as well. Experiments with the application of BioRegulators on cold stress plants have been started.
Figure 1 Abiotic stress conditions for Arabidopsis thaliana. Cold stress conditions: plants on soil are subjected to a day/night regime of 4°C. The pictures show stressed and control plants after a period of 144 hrs.Figure 2 Abiotic stress conditions for Arabidopsis thaliana. Drought stress conditions: Plants grown in hydroponic cultures are subjected to drought stress by supplementing the liquid medium with 20% PEG. The pictures show stressed and control plants after a period of 144 hrs.Figure 3 Abiotic stress conditions for Arabidopsis thaliana. Salt stress conditions: Plants grown in hydroponic cultures are subjected to salt stress by supplementing the liquid medium with 200mM NaCl. The pictures show stressed (left) and control plants (right) 144 hrs after application of salt
We have also been able to set up very reliable drought and salt stress assays for rice plants and in vitro cultures. At least two BioRegulators showed discernable effects in preliminary experiments.
Figure 4 Drought stress of rice plants (PEG 800)Figure 5 Drought stress of rape seed

 

Salt stress assays in two lettuce and three tomato cultivars have been established as well. Different growth parameters (e.g. number of leaves, biomass) and photosynthetic parameters (net CO2 fixation, stomatal conductance, chlorophyll fluorescence) are being evaluated.

The conditions for sample preparation for transcriptome analyses of Arabidopsis and rice plants have been set up. We are using the Arabidopsis ATH1 and the Rice Genome Array from Affymetrix to analyse the changes in gene expression of stressed/non-stressed plants with/without application of BioRegulators.

2D electrophoresis using fluorescent protein tags (DIGE, differential in gel electrophoresis) will be used for the identification of protein biomarkers which are indicative for BioRegulator effects in stressed plants. A first comparative study of the leaf proteome with regards to the effect of cold stress over a period of 7 days has been performed using the DIGE system. After 144 hrs of cold stress a total of 112 protein spots were found to be differentially regulated. Of these, 64 spots increased and 48 spots decreased in abundance. In total ~ 1300 spots were detected on the 2D map.
Figure 6 Comparative study of the whole leaf proteome with regards to the effect of cold stress conditions (4°C) using the 2D-DIGE system. With this method, two samples from different conditions to be compared are labelled with fluorescent dyes that are spectrally distinguishable (Cy3 and Cy5) and run on the same 2D gel. An internal standard consisting of a pool of all samples present in the experiment (labbeled with Cy2) is run on every gel in order to facilitate inter-gel matching and statistic evaluation. A. 2D image with an overlay of the images from three different dyes. B. 2D image where the protein spots that were differentially regulated in response to 144 hrs of cold stress are marked.

 

To establish a cell based screening assay for new Bioregulators an Arabidopsis cell suspension cultures has been initiated. The method for stable transformation of cells via co-cultivation with Agrobacterium tumefaciens has been used to produce cells expressing the fluorescent marker protein DsRed and HSA. Transformed cells are currently characterized with regards to their growth and protein production.

In 2008 several field trials with wheat, corn, tomato and lettuce under abiotic stress conditions are being performed to test the effect of Bioregulators in crops in the field.

Figure 7 Overview of bioregulators activities