Genetic variability and protein-protein interactions of puroindolines in relation to wheat grain texture and antimicrobial properties

The puroindoline genes, Pina-D1 and Pinb-D1, have been shown to be linked to grain hardness, an important trait in the end-use quality of wheat and hence in international usage and trade of wheat. Variations, in either or both genes, or lack of expression of either gene, result in a hard texture of common wheat, while the durum wheat that lacks both Pin genes is extremely hard. Depending on the type of mutation, a range of hardness observed. There has been a great interest to search for further genetic variations in these genes, firstly, to understand better the way these genes affect grain texture, and secondly, to introduce new variations into breeding programs in order to improve the end-use quality of wheat. Landraces of wheat, particularly from China (the secondary centre of origin of wheat), were analyzed and a new Pina-D1 allele was discovered, in addition to confirmation of several other reported alleles. Scanning electron microscopy analysis of various wheats with differences in their Pin alleles, showed more attachment between starch and protein matrix for those wheats either lacking Pins, or with potential truncation in either PIN, in comparison to the wheat with a single amino acid substitution in the Pin allele. Analysis of the Pinb-2 genes showed that these comprised a multigene family and were greatly diverse. Many new haplotypes were identified in hexaploid, tetraploid and dipoid wheats, including a new group designated Pinb-2v6. Synthetic peptides, designed based on the truncated tryptophan rich domain (TRD) of the putative PINB-2 proteins, comprising of only two Trps, showed antibacterial and antifungal activity, suggesting that the PINB-2 proteins could be of importance in seed defense, as suggested for the PINs. Many studies have suggested that the PIN proteins (PINA and PINB) were interdependent and interact to confer the grain softness trait. Examination of this aspect using the yeast two-hybrid system showed that PINA and PINB did interact physically, thus shedding some light on their possible function and supporting suggestions that they co-operatively bind to lipids. PINA was also able to interact with itself, which may be relevant to more number of Trps present at the TRD in comparison to PINB and GSP-1 which may have some association to its suggested ability to aggregate at the lipid membrane. The observed interactions of GSP-1 with PINA or PINB, suggest that the GSP-1 could be a minor factor influencing grain hardness.