Characterisation of Arabidopsis thaliana ATP Dependent Phosphofructokinases and a Putative Myristoylation Site on AtF2KP – Institut for Plante- og Miljøvidenskab - Københavns Universitet

Characterisation of Arabidopsis thaliana ATP Dependent Phosphofructokinases and a Putative Myristoylation Site on AtF2KP

PhD defence by Laura Kathrine Perby

Primary carbohydrate metabolism connects all parts of plant metabolism. When carbon
fixed via photosynthesis is used for synthesis of sucrose, or when starch is
broken down during the night and is used for e.g. sucrose synthesis,
respiration or production of defence compounds or other secondary metabolites,
it goes through core reactions in central carbohydrate metabolism.
Consequently, the core reactions of carbohydrate metabolism are crucial for
plant development and fitness upon environmental changes. One type of enzymes
that holds a key position, is the cytosolic ATP-dependent phosphofructokinases
(PFKs), of which plant typically have several isoforms. These enzymes
phosphorylate the C-1 position of Fru-6-P, leading the flux of carbon in the
glycolytic direction. Despite a central position in plant carbohydrate
metabolism, there are no reports of active plant PFK expressed in a non-plant
system. Furthermore, there are no published studies of stable transgenic plants
with altered plant PFK activity. Therefore, knowledge on the impact of PFKs on
overall carbon flux and especially on individual isoforms including their in
planta significance, is so far very limited. In this PhD study, cytosolic
A. thaliana PFKs were cloned and expressed in E. coli and initial
experiments regarding purification and enzymatic activity were carried out.
This revealed that at least one of the A. thaliana isoforms, can be purified as
an enzyme with the anticipated PFK activity. In order to study the in planta
significance of the individual cytosolic PFK isoforms, homozygous A. thaliana
single and double knock-out lines were obtained. One of the double mutants was found to have a strong growth and development phenotype. Another enzyme central to plant
carbohydrate metabolism is the bifunctional enzyme F2KP (fructose-6-phosphate
2-kinase/fructose-2,6-bisphosphatase). This enzyme is responsible for making
and degrading the signalling metabolite Fru-2,6-BP, which during photosynthesis
controls the flux of carbon into sucrose and starch synthesis. This enzyme
carries a long N-terminal extension that had so far received very little
attention. In this PhD study, a putative N-terminal myristoylation site of A.
thaliana F2KP was investigated.

Supervisor

Associate professor Tom Hamborg Nielsen

Assesment Comittee

Professor Vaughan Hurry, Umeå Plant Science Center, Sweden

Professor Kåre Lehmann Nielsen, Aalborg University, Denmark

Associate Professor Andreas Blennow (chair)

Reception afterwards in the 117 rooms