Biochemical analysis of the functionality of puroindoline peptides and proteins

The two puroindoline proteins of wheat, puroindoline-a (PINA) and puroindoline-b (PINB) have been studied extensively and their role in grain texture has been well established. Additionally, there are strong suggestions for an in vivo role for PINs in seed pathogen defence. Both PINs are required to be present together and in their wildtype functional form to exert their full 'softening' effect on the wheat grains. Absence of, or variation in, either or both PINs, result in the 'hard' grain phenotype. Yet, the effects of these hardness-associated mutations on the antimicrobial activities and the biochemical and cellular mechanisms of action remain unclear. In the present work, peptides based on the tryptophan rich-domain (TRD) of the wild-type and mutant PIN sequences, as well as barley hordoindolines (HINs) and the wheat grain softness protein-1 (GSP-1) were tested, and found to have a broad range of antibacterial and antifungal activities, suggesting this is a conserved feature of this family of proteins. Additionally, when applied exogenously, the peptides showed inhibitory activities towards leaf rust (Puccinia triticina), an important fungal pathogen of wheat. The peptides had high selectivity towards microbial cells and no toxicity towards mammalian cells, a feature which is essential for potential therapeutic applications. Scanning electron microscopy of yeast cells treated with the PINA peptide showed intact cells with pore-like structures in the membranes, suggesting a non-lytic permeabilisation mechanism. Several intracellular targets are available to non-lytic peptides once they cross the cell membrane. Inhibition of DNA synthesis was investigated, and showed the peptides have a strong DNA binding affinity and may inhibit DNA synthesis in E. coli cells. Many studies have suggested that PINs may interact co-operatively or interdependently to confer the soft grain texture. The significance of various domains and residues for any potential protein-protein interactions (PPI) was tested using the yeast-two hybrid system. Deletion of the TRD in PINA, or amino acid substitutions in it, did not adversely affect the PPI with itself and PINB, nor did the hardness-associated Leu60Pro mutation, thus these regions are unlikely to be directly involved. Deletion of the hydrophobic domain (HD) in PINA lead to a reduction in interaction, suggesting the HD is important but not essential for the PPI of PINA. The Pinb-2 genes show great diversity but may have a minor role is grain texture. Preliminary analysis of the Pinb-2 haplotypes of hard wheat cultivars with the same Ha locus genotype but variations in grain hardness suggested the Pinbii 2v3 allele could be a minor factor in increasing grain hardness. In conclusion, the PIN proteins as well as peptides have a significant potential of applications in agricultural and other industries.