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Reducing Cyanide–Dependent ROS Production in Transgenic Cassava Roots: Impact on Post-Harvest Physiological Deterioration Tawanda Zidenga1, Dimuth Siritunga2 and Richard Sayre1 1.Department of Plant Cellular and Molecular Biology, The Ohio State University 2. Department of Biology, University of Puerto Rico Post-harvest physiological deterioration is a major problem in cassava farming Deterioration occurs in 72 hrs Post-harvest physiological deterioration is a major problem in cassava farming PPD affects quality of crop for consumption and marketing There are land use implications to harvesting cassava per piece meal There are several methods for controlling PPD 1. Harvest when needed 2. Wax the roots 3. Process as soon as harvested 4. Use long shelf life cassava lines Summary Linamarin as a transportable form of reduced nitrogen in cassava The oxidative burst in wounded cassava roots is induced by cyanide release Reducing cyanide-induced ROS production via expression of Alternate oxidase Reducing cyanide-induced ROS may be a strategy to control postharvest physiological deterioration in cassava PPD is a biochemically active process PPD is associated with an oxidative burst in cassava roots There is also production of phenolic compounds like scopoletin And, increase ethylene biosynthesis Cyanide release in higher plants Ethylene Biosynthesis methionine S-adenosyl methione Degradation of cyanogenic glucosides Linamarin Acetone cyanohydrin ACC Acetone + HCN Ethylene + HCN + CO2 + H2O All plants Cyanogenic plants Linamarin is made in the leaves and transported to the roots Linamarin Unprocessed cassava releases about 53 mg HCN per 100g fresh weight. Minimal lethal dose of HCN 0.5-3.5mg per kg body weight. The fate of cyanide in the root may have important implications on single nitrogen metabolism in cassava DETOXIFICATION Rhodanese Thiocyanate ASSIMILATION β-Cyanoalanine synthase Amino acids, Ammonia Hypothesis: Assimilatory pathway, which incorporates cyanide into nitrogen metabolism, is preferred in cassava roots Rhodanese is barely detectable in cassava roots Leaf Activities Root Activities Root β-cyanoalanine synthase activity is 3X the shoot activity Cyanogenic glucosides are assimilated via βCyanoalanine synthase in cassava roots ASSIMILATION β-Cyanoalanine synthase Amino acids, Ammonia Over-expression of Nitrilase 4 and β-cyanoalanine synthase to enhance assimilation of cyanide in cassava roots ROOT Linamarin Hypothesis: Root-specific expression of βCAS and NIT4 will increase amino acid pool sizes in cassava roots and reduce cyanideinduced ROS production Linamarase Acetone cyanohydrin HNL Cyanide + Cysteine β-cyanoalanine synthase Cyanoalanine + H2O Nitrilase/hydrase Asparagine Aspartate + NH3 Current and future work on cyanide assimilation Screening NIT4 and CAS plants Analysis of amino acid pool sizes in the transgenics compared to wild type plants Analysis of levels of linamarin in transgenics compared to wild type plants Using Low Linamarin transgenics to understand cyanide metabolism in cassava roots 100 99 98 97 96 3.40 3.36 3.32 3.28 % Linamarin 0.08 2.0 0.98 0.73 0.06 1.5 0.04 1.0 0.29 0.5 0.33 0.02 0.26 0.0 0.00 WT Siritunga and Sayre, 2003 Cab1-1 Cab1-2 Cab1-3 Cab1-4 Cab1-5 Linamarin (umoles/gdw) 2.5 Low cyanide cassava roots are significantly smaller CYP79D1/CYP79D2 antisense with PATATIN promoter CYP79D1/CYP79D2 antisense with CAB promoter P8 C3-12 P6 C3-5 P4 C3-1 Cyanide is an inhibitor of cytochrome oxidase in mitochondrial respiratory chain Cyanide sensitive Biochemistry and Molecular Biology of Plants Tissue disruption in cassava releases cyanide Linamarin Linamarase Acetone cyanohydrin HNL Acetone + Cyanide Analyzing ROS production in low and high cyanide cassava • Analysis of reactive oxygen species production in wild type and low cyanide cassava using fluorescent probe • Root-specific expression of Alternative oxidase as a strategy to control PPD in cassava Hypothesis: Cyanide initiates post-harvest physiological deterioration through increased reactive oxygen species production Over-expression of Nitrilase 4 and β-cyanoalanine synthase to enhance assimilation of cyanide in cassava roots ROOT Linamarin Hypothesis: Root-specific expression of βCAS and NIT4 will increase amino acid pool sizes in cassava roots and reduce cyanideinduced ROS production Linamarase Acetone cyanohydrin HNL Cyanide + Cysteine β-cyanoalanine synthase Cyanoalanine + H2O Nitrilase/hydrase Asparagine Aspartate + NH3 Plants over-expressing Nitrilase 4 show reduced production of ROS NIT1 NIT1 NIT1 60444 60444 60444 The oxidative burst in wounded cassava roots is cyanogen induced CONTROL (no dye) CAB (low cyanide) WILD TYPE Mean 18 16 14 12 10 Mean 8 6 4 2 0 Control Low cyanide Wild Type Cyanide complements ROS production in low cyanide plants CAB (low cyanide) WILD TYPE CAB + 5mM NaCN Mean 25 20 15 Mean 10 5 0 Low cyanide Wild Type Low cyanide + 5mM NaCN The ROS-induced fluorescence is mitochondrial WILD TYPE WILD TYPE + DPI Mean . 20 18 16 14 12 10 8 6 4 2 0 Mean Wild Type Wild type + DPI The NADPH oxidase inhibitor, diphenyl iodonium chloride (DPI), does not inhibit ROS production in wild type cassava roots Possible strategy to reduced cyanide-induced ROS – express Alternative oxidase Cyanide insensitive Biochemistry and Molecular Biology of Plants Cyanide sensitive Why Aox • Overproduction of AOX in transgenic cell lines reduces ROS production • Antisense cells with reduced levels of the AOX accumulate five times more ROS than control cells Annu. Rev. Plant Biol. 2004. 55:373–99 Alternative oxidase reduces ROS production in cassava roots Wild type AOX-3 AOX-1 18 16 14 12 10 8 6 4 2 0 60444 AOX1 AOX3 Current and future work on cyanide, ROS and PPD Generating more transgenics Evaluating confirmed transgenics for ROS Transferring transgenics to Greenhouse Evaluating AOX transgenics for PPD Evaluating low cyanide plants for PPD Summary of strategies for controlling PPD ROS scavenging enzymes Amino acids, NH3 ROS Cyt ox Assimilation PPD HCN 2 Cyanide 3 assimilation Cyanide elimination Aox 1 Sayre Lab Cassava Group Dr. Uzoma Ihemere Dr. Narayanan N. Narayanan Dr. Hangsik Moon Elisa Leyva –Guerero Anthonia Soboyejo Reid Rice Sayre Lab Chlamy Group Dr Shayani Dr Vanessa Falcao Anil Kumar Zoe Gokhale Dr. Mary-Ann Abiado Ohio State University Microscopy Core Lab Facility Biao Ding Microscopy Lab BioCassava Plus