The non-specific lipid transfer proteins (nsLTPs) are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine: (1) when the nsLTP family arose, (2) how often new nsLTP subfamilies have been created, and (3) how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system.

Several families of G protein-coupled receptors (GPCRs) show no significant sequence similarities to each other, and it has been debated which of them share a common origin. We developed and performed integrated and independent HHsearch, Needleman--Wunsch-based and motif analyses on more than 6,600 unique GPCRs from 12 species. Moreover, we mined the evolutionary important Trichoplax adhaerens, Nematostella vectensis, Thalassiosira pseudonana, and Strongylocentrotus purpuratus genomes, revealing remarkably rich vertebrate-like GPCR repertoires already in the early Metazoan species. We found strong evidence that the Adhesion and Frizzled families are children to the cyclic AMP (cAMP) family with HHsearch homology probabilities of 99.8% and 99.4%, respectively, also supported by the Needleman--Wunsch analysis and several motifs. We also found that the large Rhodopsin family is likely a child of the cAMP family with an HHsearch homology probability of 99.4% and conserved motifs. Therefore, we suggest that the Adhesion and Frizzled families originated from the cAMP family in an event close to that which gave rise to the Rhodopsin family. We also found convincing evidence that the Rhodopsin family is parent to the important sensory families; Taste 2 and Vomeronasal type 1 as well as the Nematode chemoreceptor families. The insect odorant, gustatory, and Trehalose receptors, frequently referred to as GPCRs, form a separate cluster without relationship to the other families, and we propose, based on these and others' results, that these families are ligand-gated ion channels rather than GPCRs. Overall, we suggest common descent of at least 97% of the GPCRs sequences found in humans.

The non-specific lipid transfer proteins (nsLTPs) constitute a large protein family specific for plants. Proteins from the family are found in all land plants but have not been identified in green algae. Their in vivo functions are still disputed although evidence is accumulating for a role of these proteins in cuticle development. In a previous study, we performed a co-expression analysis of glycosylphosphatidylinositol (GPI)-anchored nsLTPs (LTPGs), which suggested that these proteins are also involved in the accumulation of suberin and sporopollenin. Here, we follow up the previous co-expression study by characterizing the phenotypes of Arabidopsis thaliana lines with insertions in LTPG genes. The observed phenotypes include an inability to limit tetrazolium salt uptake in seeds, development of hair-like structures on seeds, altered pollen morphologies and decreased levels of ω-hydroxy fatty acids in seed coats. The observed phenotypes give further support for a role in suberin and sporopollenin biosynthesis or deposition in A. thaliana.

The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing.

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