Plant lipid transfer proteins : Evolution, expression and function

Sammanfattning: The plant non-specific lipid transfer proteins (nsLTPs) are known for the ability to transfer different lipids in vitro, but their in vivo functions have not yet been elucidated. They seem to play a role in the defense against biotic and abiotic stresses; the gene expression of nsLTPs is often upregulated when exposed to stresses. Further, two different nsLTPs have been shown to affect the lipid composition of the plant cuticle, a structure acting as a protective barrier. However, more evidence is needed to prove this hypothesis and to pinpoint their exact role in this process.In this thesis I have shown that the nsLTPs are found in all land plants, but not in any of the studied algae. This supports a role in defense response, since protection against dehydration, radiation, pathogens and other stresses played a crucial role when plants adapted to a life on land. Characterization of the nsLTPs in early diverging land plant revealed that even though the amino acid similarity towards nsLTPs in flowering plants is not very high, the main properties of the proteins are still the same (Paper I). This includes the protein structure, which consists of α-helices surrounding a lipid binding cavity, a conserved pattern of cysteine residues involved in disulphide bonds and a signal sequence directing the protein to the  extracellular space. Further, the expression of nsLTPs in the moss Physcomitrella patens was shown to respond to stresses, and construction of an YFP-LTP fusion protein confirmed the localization to the periphery of the cell in planta (Paper II). Heterologous expressed Physcomitrella nsLTPs were also shown to have the ability to bind lipids and to be very heat stable, features previously only studied in nsLTPs from flowering plants. By examining the presence of a cuticle in Physcomitrella, a correlation between the nsLTPs´ lipid binding ability and the lipid composition of the cuticle could be found, which further strengthens the involvement of nsLTPs in transfer of lipids for cuticle construction.In the flowering plant Arabidopsis thaliana, I showed that several of the nsLTPs followed the same expression pattern when examining data from different tissues, stress treatments, hormones, chemical treatments and developmental stages, but also that four of the genes were undergoing alternative splicing resulting in different isoforms of the proteins (Paper III). Based on their expression patterns, the genes could be divided into three different coexpression networks. By examining other genes similarly expressed, each network could be designated to a putative function: Transfer of lipids for synthesis of the cuticle, suberin layer and sporopollenin, respectively. In Paper IV, these hypotheses were tested in vivo by examining knockout mutants of several nsLTPs in Arabidopsis. The involvement in sporopollenin deposition could be confirmed; two of the knockout lines showed collapsed pollen grains. Further, two other lines showed an increased seed coat permeability due to an altered lipid composition of the suberin layer. Together, the results support a role for nsLTPs in construction of the protecting barriers in all land plants.