PI: Tuula Teeri

Staff: Alex Rajangam, PhD student

Collaborations: Ines Ezcurra, docent KTH; Christina Divne, docent KTH; Hao Hu, PhD student KTH

Background

Expression profiling using microarrays has been carried out previously in order to identify genes involved in the secondary cell wall formation in the hybrid aspen, Populus tremula L. x tremuloides Michx. (Aspeborg et al. 2005). In addition to genes with predictable functions, many genes were identified with previously unknown functions. A gene with one of the highest relative levels of expression during early secondary cell wall synthesis was denoted PttMAP20. This gene encodes a 20.7-kDa protein, which contains a highly conserved domain present in the TPX2 protein mediating the binding of a kinesin-like protein, Xklp₂, to microtubules in Xenopus. By using software recognizing distant homologous relationships between proteins PttMAP20 was found to bear structural similarity to stathmins, which are involved in regulating the dynamic stability of vertebrate microtubulins. Immunolocalization studies relying on antibodies raised against recombinant PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell wall. Within the xylem cell, the protein resides in the periphery of the living protoplast close to the plasma membrane where also cortical microtubules are found. Both native and recombinant PttMAP20 were shown to bind to in vitro assembled, taxol stabilized mammalian and hybrid aspen microtubules. The data obtained show that PttMAP20 is a novel microtubule associated protein in the wood forming tissues of hybrid aspen. Since microtubules apparently have a role in the organized assembly of cellulose microfibrils, microtubule-associated proteins are interesting objects for studies of cell wall assembly.forming secondary cell wall. Within the xylem cell, the protein resides in the periphery of the living protoplast close to the plasma membrane where also cortical microtubules are found. Both native and recombinant PttMAP20 were shown to bind to in vitro assembled, taxol stabilized mammalian and hybrid aspen microtubules. The data obtained show that PttMAP20 is a novel microtubule associated protein in the wood forming tissues of hybrid aspen. Since microtubules apparently have a role in the organized assembly of cellulose microfibrils, microtubule-associated proteins are interesting objects for studies of cell wall assembly.

Aims

The aim of the present project is to investigate the role in wood development and the molecular function on PttMAP20 and other putative microtubule associated proteins identified in poplar xylem. These studies will involve the identification of the microtubule-binding domain of MAP20, investigation whether phosphorylation regulates the function of MAP20, determination of the three-dimensional structure of MAP20, complementation studies of KO-mutants of putative orthologs of PttMAP20 in Arabidopsis and investigations of the effects of RNAi down-regulation of the PttMAP20 gene in poplar. The experiments with Arabidopsis and poplar will be carried out in collaboration with Björn Sundberg and Ewa Mellerowicz.

References

• Aspeborg H, Schrader J, Coutinho PM, Stam M, Kallas Å, Djerbi S, Nilsson P, Denman S, Amini B, Sterky F, Master E, Sandberg G, Mellerowicz E, Sundberg B, Henrissat B, Teeri TT (2005) Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen. Plant Physiol 137: 983-997