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3. Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production1[C][W][OA]

Author

Zidenga, Tawanda, Leyva-Guerrero, Elisa, Moon, Hangsik, Siritunga, Dimuth, and Sayre, Richard

Subjects
Manihot, Plant Stems, Biochemical Processes and Macromolecular Structures, Arabidopsis, food and beverages, Hydrogen Peroxide, Plants, Genetically Modified, Models, Biological, Plant Roots, Mitochondria, Mitochondrial Proteins, Plant Tubers, Phenotype, Nitriles, Biomass, Oxidoreductases, Reactive Oxygen Species, Plant Proteins, Respiratory Burst
Abstract

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.

Published
2012

6. Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production1[C][W][OA].

Author

Zidenga, Tawanda, Leyva-Guerrero, Elisa, Moon, Hangsik, Siritunga, Dimuth, and Sayre, Richard

Subjects
*CASSAVA research, *REACTIVE oxygen species, *POSTHARVEST diseases, *ARABIDOPSIS thaliana, *OXIDATIVE stress, *PHYSIOLOGY
Abstract

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanideinduced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration. [ABSTRACT FROM AUTHOR]

Published
2012
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7. Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production1[C][W][OA].

Author

Zidenga, Tawanda, Leyva-Guerrero, Elisa, Moon, Hangsik, Siritunga, Dimuth, and Sayre, Richard

Subjects
CASSAVA research, REACTIVE oxygen species, POSTHARVEST diseases, ARABIDOPSIS thaliana, OXIDATIVE stress, PHYSIOLOGY
Abstract

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanideinduced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration. [ABSTRACT FROM AUTHOR]

Published
2012
Full Text
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8. Enhancement of the free amino acid and protein content of cassava storage roots and evaluation of root-specific promoters in cassava

Author

Leyva-Guerrero, Elisa

Subjects
Botany, Plant Biology, cassava, free amino acid increase, protein increase, cyanogenic glucosides, linamarin, promoters, nitrate reductase, linamarase, sporazein, storage root
Abstract

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.

Published
2011

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