ADAPTABILITY Project Description
Workpackage 1
Workpackage 1.1
Workpackage 1.2
Workpackage 1.3
Workpackage 1.4
Workpackage 1.5

Workpackage 2
Workpackage 2.1
Workpackage 2.2
Workpackage 2.3

Workpackage 3
Workpackage 3.1
Workpackage 3.2

Workpackage 4
Workpackage 4.1
Workpackage 3.1.
Environmental effects on methylation of DNA and translation of phytochrome genes
Start date: 1st month Partners responsible: NISK
Completion date: 36th month Other partners involved: TUM
  1. To determine if the treatments with light and temperature on plants of Norway spruce will affect the general methylation pattern of its DNA in vegetative buds
  2. To determine if the above mentioned treatments affect the levels of RNA encoding different phytochromes during the process of cold acclimation
  3. To determine if the localisation pattern and pattern of accumulation in vegetative buds of the two known phytochromes is affected by the different treatments of the plants
Description on the work
For objective 1, DNA is extracted from seedlings and small spruce plants with clear differences in adaptation to cold and light treatment. Since it is well known that environmental factors affects the methylation of DNA the extracted DNA will be digested with methylation sensitive and insensitive restriction enzyme isoschizomers, amplified with a PCR technique described for this type of analyses, and analysed using agarose gel electrophoresis. Several previous results suggest that phytochrome may be a key factor in the adaptation process. Two different cDNA's encoding phytochromes has been isolated from Norway spruce. These will be used for objective 2, to follow the expression of the corresponding genes during bud set in RNA hybridisation experiments. The group has experience to isolate high quality RNA from different tissues of this recalcitrant plant and has also equipment to quantify the signals. To achieve objective 3 the sequence of the two phytochrome cDNA’s will be utilised to design a peptide to be used to generate specific antibodies. These antibodies is then used in immunolocalization experiments to follow the localisation pattern and pattern of accumulation during the development of vegetative buds
Two papers in international scientific journals with referees will be published from this part of the project. One will deal with the DNA methylation and the other will deal with the effects on transcription and translation of the two phytochrome genes; 0-36
1° year:
  • Extraction of DNA from vegetative buds of plants differentially adapted, and initiation of methylation analysis
  • Extraction of RNA from vegetative buds of plants differentially adapted
  • Synthesis of phytochrome peptides and generation of antibodies against these peptides

2° year:

  • Continued analysis of DNA methylation, including digestion and selective PCR amplification
  • Analysis of phytochrome transcript accumulation during development of vegetative buds using RNA hybridisation
  • Antibodies against two phytochromes generated and specificity determined

3° year:

  • DNA methylation analysis finished and the results are analysed
  • Analysis of phytochrome transcript finished and analysed
  • Immunolocalization of phytochromes finished and analysed
  • Two manuscripts describing the results are written and submitted to journals
© 2001 ISOGEN