- ItemTowards the Identification and Isolation of VvPNP(Stellenbosch : University of Stellenbosch, 2008-03) Kerr, Mark Stuart; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.We have identified a novel plant natriuretic peptide (PNP) like gene in Vitis vinifera cultivars of Chardonnay and Pinotage. The transcript of the gene was isolated from young leaves indicating that the peptide may function within these organs of the plant. The 15 kDa peptide, which we have putatively called Vitis vinifera plant natriuretic peptide (VvPNP), was expressed in a bacterial system as a recombinant fusion protein. This protein shares sequence similarity to other recognised natriuretic peptides, and was largely identified by using the primary sequence of the well characterised plant natriuretic peptide (AtPNP-A) from the model organism Arabidopsis thaliana. Previous studies have identified PNPs across a range of plant species such as Dracena godseffiana, Hedera helix and Solanum tuberosum, where the biological activity associated with water and solute homeostasis has been proven. Instrumental to the functionality of these peptides is the conservation of two cysteine residues which form a disulphide bridge, of no less than 23 amino acids apart, creating a secondary ring structure. Alignment of the VvPNP and AtPNP-A primary structures indicates that the two cysteine residues necessary for physiological function in AtPNP-A are in conserved positions within VvPNP. Also 14 identical amino acids and 7 conservative amino acids aligned within the active domain of the AtPNP-A molecule. The putative natriuretic peptide also displays two diagnostic amino acids motifs characteristic in PNPs and one other molecule CjBAp12, which is associated with citrus blight. On a nucleotide level the VvPNP contains a 100bp intron which is also found within the genomic sequence of AtPNP-A. Plant natriuretic peptides have sequence similarities to expansins, molecules which directly modify the mechanical properties of cell walls leading to turgor-driven cell extension. Although PNPs do not contain a wall-binding domain, evidence does suggest an evolutionary relationship between expansins and PNP molecules because of these similarities. The target site for PNPs being the cell membrane and not the cell wall, and it is for this reason that PNPs affect protoplasts. Although VvPNP shares some similarity with the domain organisation of expansins it lacks the tryptophan rich C-terminal domain. This domain also makes expansins larger ( 25 kDa) than natriuretic peptides ( 14 kDa). Attempts to show functionality of the recombinant fusion protein GST:VvPNP were unsuccessful thus far. Further inquiries into the role that VvPNP plays in the homeostasis of grapevine are needed to elucidate the potential enhancement for this important economic crop.