In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

Abstract

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F 2 :F 3 populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophanrich region is the most important region of both PINA and PINB.

Characterization and sequence diversity of the Gsp-1 gene in diploid species of the Aegilops genus

“…Consequently, this protein is likely to be less stable than the others and therefore could affect grain hardness. In fact, similar changes have been found in PINs resulting in increased hardness (Feiz et al, 2009). To date, mutations at cysteine level in GSP-1 have only been detected by Wilkinson et al (2013) but not in Aegilops.…”

“…The Trp-domain has been shown to be the most important region of both PINA and PINB, being essential to preserve the softness texture of grain (Feiz et al, 2009). The hydrophobic Trp residues show an affinity for polar lipids on the surface of starch granules (Pauly et al, 2013).…”

“…Although the main determinant of grain texture has been associated with the presence/absence or modification of Pin genes (Bhave and Morris, 2008;Feiz et al, 2009), some studies have suggested that the Gsp-1 gene could also have a role in this important grain trait (Morris et al, 2013). Although Tranquilli et al (2002) found that deletions or allelic variants of Gsp-A1 and Gsp-B1 had no significant impact on grain texture, Gedye et al (2004) showed that a significant amount of variation in hardness of synthetic wheat was assignable to the durum wheat parent and suggested a role for GSP-1 and other QTLs associated with the hardness.…”

Prevalence of Puroindoline D1 and Puroindoline b-2 variants in U.S. Pacific Northwest wheat breeding germplasm pools, and their association with kernel texture

“…The Puroindoline D1 genes have a dramatic impact on kernel texture, a significant effect on processing quality and may play a role in plant disease defense (Chen et al 2007;Feiz et al 2009;Morris 1997, 1998;Lillemo and Morris 2000;Ma et al 2009;Zhang et al 2010Zhang et al , 2011 Morris et al 2001;Xia et al 2005). In this study, we analyzed the frequency of PinaD1b and Pinb-D1b among four hard wheat groups (hard red winter, hard white winter, hard red spring and hard white spring), and found that 94 of 101 hard winter wheats (red and white) carried Pina-D1b (Table 3).…”

Variability in yield traits of TILLING population of bread wheat (Triticum aestivum L.)

“…Mutagenesis is an important tool in crop improvement to generate a vast amount of genetic variability for identification of improved or novel phenotypes that can be exploited in conventional plant breeding programmes and genetic studies (Parry et al, 2009). Induced mutations through non transgenic technique called Targeting Induced Local Lesions IN Genomes (TILLING) are now being widely used for developing improved crop varieties and for the discovery of genes controlling important traits and understanding the functions and mechanisms of actions of these genes in wheat Dong et al, 2009;Uauy et al, 2009;Feiz et al, 2009;. The range of alleles can be developed in a short time is matchless and unlikely to be found elsewhere in the pool of germplasm accessible to plant breeders, including land races and undomesticated relatives.…”