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Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava

Leyva-Guerrero, Elisa
http://rave.ohiolink.edu/etdc/view?acc_num=osu1299535104

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Plant Cellular and Molecular Biology.
Cassava is an important staple crop for millions of people around the world in particular in Sub-Saharan Africa. Cassava storage roots are a good source of calories, however, they are deficient in protein and other micronutrients such as iron and zinc. Protein malnutrition is widespread in the regions were cassava is widely consumed and consumption of cassava as a staple has been linked to reduced protein intake in the diet. A cassava storage root with higher protein could potentially impact the nutrition and well being of millions people. In this thesis we propose two approaches for increasing free amino acids and protein in cassava storage roots. One approach was to increase nitrate assimilation through the expression of a mutated nitrate reductase. Nitrate reduction to nitrite is followed by a further reduction to ammonium which can be readily incorporated into amino acids. A rate limiting step in nitrate metabolism is the reduction of nitrate to nitrite by nitrate reductase. This enzyme is highly regulated, however, it has been observed that through mutation of a key regulatory serine the enzyme remains active and the assimilation of nitrate increases resulting in increased free amino acid levels. In cassava the root-specific expression of a mutated nitrate reductase resulted in a doubling of the storage root free amino acid content; however, no root protein increase was observed. A second approach was to utilize the nitrile group present in the cassava cyanogenic glucoside linamarin as a reduced nitrogen source. The hydrolysis of linamarin by the enzyme linamarase releases acetone cyanohydrin which in turn can degrade to release cyanide. In all plants there is a cyanide assimilation pathway involving β-cyanoalanine synthase, the end products of this pathway are aspartate and ammonia. Through increased hydrolysis of linamarin, we sought to increase the assimilation of cyanide in to aspartate and ammonia. In wild-type cassava the interaction between linamarase and linamarin is limited spatially; linamarase is found in the cell wall and linamarin in the vacuole. Through the expression of a vacuolar targeted linamarase, we proposed to increase the hydrolysis of linamarin and as a result provide more reduced nitrogen (nitrile) for free amino acid and protein synthesis. The expression of a vacuolar linamarase in cassava storage roots resulted in doubling of the free amino acid pool in this organ but no increase in protein. It was only through the dual expression of vacuolar linamarase and the storage protein sporazein that both an increase in free amino acid and protein in the storage roots was observed. Although cassava is a main staple food the molecular biology tools available for its transformation are very limited. In the final chapter of this thesis we evaluated four different Arabidopsis root promoters in cassava to determine their functionality and tissue specificity. Two promoters named A14 and E40 were determined to be functional in cassava roots with minimal leaf expression. The availability of an increased set of root promoters for cassava may increase the development of transgenic cassava with increased nutritional and industrial value.
Patrice Hamel (Advisor)
Richard Sayre (Advisor)
James Metzger (Committee Member)
Dave Somers (Committee Member)
Randall Scholl (Committee Member)
394 p.
Botany; Plant Biology
cassava; free amino acid increase; protein increase; cyanogenic glucosides; linamarin; promoters; nitrate reductase; linamarase; sporazein; storage root

Recommended Citations

Citations

  • Leyva-Guerrero, E. (2011). Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299535104

    APA Style (7th edition)

  • Leyva-Guerrero, Elisa. Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1299535104.

    MLA Style (8th edition)

  • Leyva-Guerrero, Elisa. "Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299535104

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.