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Title: In Planta mutagenesis determines the functional regions of puroindoline proteins.

Author
item FEIZ, LELIA - Montana State University
item MARTIN, JACK - Montana State University
item Beecher, Brian
item GIROUX, MICHAEL - Montana State University

Submitted to: PLoS Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2009
Publication Date: 9/14/2009
Citation: Feiz, L., Martin, J., Beecher, B.S., Giroux, M. 2009. In Planta mutagenesis determines the functional regions of puroindoline proteins. PLoS Genetics 183:853-860.

Interpretive Summary: Wheat grain is classified and marketed as either being of hard or soft kernel texture. Soft kernel texture is the result of the action of two wheat proteins, PINA and PINB. Loss of function in either PINA or PINB results in hard kernel texture. In this study, a soft wheat was mutagenized and 43 independent mutants in the PINA or PINB encoding genes were generated. The effect of the various mutations upon kernel texture was measured. As a result, the regions of the PINA and PINB proteins which are important to their function in controlling wheat kernel texture were identified. The most critical region appears to be that immediately surrounding a cluster of Tryptophan amino acid residues. In addition, it was found that the function of the PINB protein is more easily disrupted than that of PINA.

Technical Abstract: PINA and PINB functionally comprise the wheat (Triticum aestivum L.) Ha locus which controls grain texture and many wheat end-use properties. PINs in their functional forms impart soft texture and a mutation in either PIN is found in all hard wheats. Studies of the PINs mode of action is limited by low allelic variation and tight linkage. To create new Pin alleles and identify critical regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 43 unique PIN missense alleles was then measured using segregating F2:F3 populations. The impact of missense alleles upon PIN function as measured by grain hardness increases ranged from neutral (72 %) to intermediate (11 %) to function-abolishing (19 %). 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 locus function. This is contrary to expectations in that PINB is not as well conserved among wheat relative species as PINA. Region by region analysis demonstrated that all function abolishing mutations resulted from structure disrupting mutations (cysteine substitutions) or from mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.