Photosynthesis

A basic property of plants is their ability to carry out photosynthesis and thus to provide the organic carbon which forms the basis for all our food, feed and biomass. The photochemical reactions of photosynthesis proceeds in the thylakoid membranes of the chloroplasts and are mediated by two photosystems: PS I and PS II, catalyzing light driven electron transport across the thylakoid membrane. We have isolated PSI as a photochemically active pigment-protein complex containing 18 different subunits and we were first to discover many of these subunits and their corresponding genes. Using these tools we have studied the structure, function and biogenesis of the PS I complex. The subunits binding the redox active components have been identified. Functional studies have been carried out with isolated in vitro systems using e.g. chemical cross-linking techniques and reconstitution with heterologously expressed subunits. Functionally, PS I operates mostly in series with PS II. However, a fraction of the electrons cycle around PS I. This permits the plant to optimize the ratio between ATP and NADPH production. More importantly, the cyclic electron flow serves to control pH in the thylakoid lumen and thereby to regulate PSII activity. The tight regulation of electron transport ensures that damaging overreduction of the photosynthetic apparatus does not take place even under conditions of restricted carbon dioxide assimilation, e.g. during drought or low temperature.
 
 

Research plan:
 
Suppression of PS I subunits in transgenic plants
Model plants like Arabidopsis and tobacco are transformed with antisense and sense constructs in order to suppress the expression of specific PS I genes. Alternatively knock-out line are constructed. Most of the subunits can be suppressed without lethal effects on the plants. Most subunits are not directly involved in electron transfer and have regulatory functions that cannot adequately be studied in isolated systems. Using transgenic plants with reduced levels of specific PSI subunits, the significance and function of these subunits are studied at the whole plant level under different growth conditions. Plants lacking PSI-N, PSI-H, PSI-F, PSI-K, PSI-G, PSI-L, PSI-O, PSI-E, PSI-D and PSI-J have so far been produced and characterized at the biochemical and whole plant level. The chloroplast encoded subunits PSI-A, -B, and -C are essential and a knock-out plant mutant will not be viable. We are currently investigating the function of the recently discovered PSI-P subunit.
 

Arabidopsis plants without subunit PSI-F are severely affected in growth. The additional lack of PSI-N has no additive effect.
 
 

 

 

 

 

 

  

Assembly
We are using PS I as a model for the assembly of multi-protein complexes. Currently we use radioactive labeling of the PS I complex during biogenesis or turnover. Mutants lacking specific PS I subunits that affect the stability of the PSI are being used to delineate the biogenesis process. Likewise mutants putatively affected in assembly of the photosynthetic complexes are being characterized. One unsolved question is how the assembly process of this multi-subunit complex is coordinated with chlorophyll biosynthesis and insertion of the pigments into the proteins and complexes.

 

Import and mechanism of insertion
Most proteins localized in the chloroplast are encoded by the nucleus. This means that after translation in the cytosol the proteins are imported into the chloroplast. We have used import assays in which precursor proteins are synthesized in vitro under incorporation of a radioactive label and then mixed with intact chloroplasts. Subsequent biochemical fractionation of the chloroplast compartments (stroma and thylakoids) in combination with protease treatment of the samples can then be used to determine the localization and topology of the protein within the thylakoid membrane. With this system we have investigated the topology of some of the peripheral PS I subunits and furthermore unraveled their mechanism of insertion. This has revealed that PSI-G, which is a small protein with only two membrane spanning helices, has a stromal loop that is unusually in-sensitive to protease digestion probably because it is protected by interaction with the PSI core protein PSI-B.

Specific protein tags can be very useful to have in a large protein complex. The tags can be exploited for rapid purification, for specific detection and localization of a protein within a complex and furthermore the tag can be used to fix a complex to a solid support which is being exploited in various nanotechnology applications. We have introduced His- or Strep-tags into the PSI-G subunit and transformed plants with genes encoding the tagged versions of PSI-G. Future work will exploit the tags for purification of complete and partial assembled PSI complexes.

 

Reactive oxygen, stress and moss
In recent year a new model plant species, Physcomitrella patens, has emerged. Physcomitrella diverged from higher plants more than 450 million years ago and is highly tolerant to a range of abiotic stresses. Identifying the underlying mechanisms giving this tolerance could supply valuable information on how to improve stress tolerance of vascular plants. In moss several novel genes involved in abiotic stress protection have been identified. These genes have apparently been lost during evolution in time periods where they had no selective advantage; however, these ancient genes could however hold solutions for some of problems faced by modern agriculture.

Unlike any vascular plants known to date Physcomitrella is able to perform homologous recombination with high efficiency. In addition, the vegetative state of Physcomitrella is haploid and each cell maintains in ability to generate new gametophytes. These features render Physcomitrella a highly efficient model plant in molecular studies and allow the generation of predictable and specific gene knock-outs in one step without the need for whole plant regeneration and crossing. The full genome sequence of Physcomitrella is also available.

 

Chlorophyll biosynthesis
We have identified and characterized mutants affected in chlorophyll biosynthesis. Especially the enzyme which forms the fifth ring of the chlorophyll molecule, the cyclase, has been under investigation. This step is catalyzed, at least in part, by a diiron enzyme CHL27/CRD1.

Genetic evidence suggests that there is more than one protein involved in the cyclase step and currently we are exploiting different routes to identify the remaining subunits. Chlorophyll biosynthesis is also interesting because a tight control between pigment biosynthesis and synthesis of proteins, like the photosynthetic reaction centers and light-harvesting complexes, is expected. The involvement of Chl-biosynthesis intermediates in chloroplast signaling is appealing since it potentially coordinates synthesis of the photo-labile pigments with expression of genes encoding subunits of the pigment-binding photosynthetic complexes. However, very little is known about the signaling components and how the signal is communicated to the nucleus.

 
Head of research group:
Poul Erik Jensen
Associate professor

Tel: +45 35 33 33 40
 
Senior group members:
Christina Lunde
Associate professor

Tel: +45 35 33 33 17

 

Anna Haldrup
Department Head

Tel: +45 35 33 33 68
 
 
Group Members:
Anastassia  Khrouchtchova, post-doc
Damian Drew, post-doc
Irina Tolstygina, post-doc
Anne Stenbæk, PhD-student
Marta Powikrowska, PhD-student
Daniele Silvestro, PhD-student
 
Eva Søgaard, technician
Cherry Nielsen, technician