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, 159 (4), 1396-407

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

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Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

Tawanda Zidenga et al. Plant Physiol.

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.

Figures

Figure 1.
Figure 1.
ROS production is reduced in low-cyanogen cassava plants. ROS production is shown in high-cyanogen (wild type) and low-cyanogen (Cab1-1 to -1-3) plants. ROS accumulation is reduced in low-cyanogen (Cab1) transgenic cassava lines. A, Hydrogen peroxide (H2O2) accumulation was determined by staining with DAB and detected microscopically. B, ROS accumulation was detected using the fluorescent dye H2DCF-DA and imaged using a Zeiss LSM 510 laser confocal microscope. All transgenic low-cyanogen cassava lines (Cab1-1, Cab1-2, and Cab1-3) had significantly lower levels of ROS accumulation at P ≤ 0.05. Statistical analysis was carried out by one-way ANOVA with Dunnett’s multiple comparison test.
Figure 2.
Figure 2.
Cyanogenesis induces ROS accumulation in cassava roots. A, Biochemical complementation of low-cyanide plants with 5 mm potassium cyanide (KCN) results in increased ROS production in 4-week-old in vitro, low-cyanogen (Cab1-1 to -1-3) transgenic plants. In vitro roots were stained with H2DCF-DA and analyzed by laser confocal microscopy. Quantification of fluorescence was done using ImageJ image processing software. The data are averages of four experiments. Error bars show 95% confidence intervals. B, Inhibition of the plasma membrane NADPH oxidase. DPI (100 µm), an inhibitor of the plasma membrane NADPH oxidase, does not substantially reduce ROS production in 4-week-old in vitro cassava, suggesting that the ROS may be of mitochondrial origin. Fluorescence intensity was scored from images of three experiments using ImageJ. The data were analyzed by t tests using GraphPad Prism software (version 5). There was no significant difference between treated and untreated roots in the treatment in B at P ≤ 0.05. Error bars show confidence intervals at P ≤ 0.05. [See online article for color version of this figure.]
Figure 3.
Figure 3.
Expression of Arabidopsis AOX in greenhouse-grown transgenic cassava roots. A, Plasmid construct of AOX. The codon-optimized Arabidopsis AOX, AtAox1A, was cloned into a pBI121-based three-dimensional vector in which the cauliflower mosaic virus 35S promoter was replaced by the root-specific patatin promoter followed by the NOS terminator (Siritunga and Sayre, 2003; Ihemere et al., 2006). B, AOX expression in roots of transgenic lines as determined by RT-PCR. RNA was extracted from 4-week-old in vitro lines. Primers specific to the end of AOX1A and the beginning of the NOS terminator were used to verify the presence of transgene. Expression was normalized by α-tubulin primers. The expected 500-bp band for the AOX transgene was seen in PAOX1 to -7 but not in the wild type (WT). C, AOX activity in roots of wild-type and transgenic plants overexpressing AOX. The data (nmol oxygen mg−1 protein min−1) are averages of three experiments. Data analysis was by one-way ANOVA with Dunnett’s multiple comparison test. Error bars show 95% confidence intervals. All transgenic lines were significantly different from the wild type at P ≤ 0.05. [See online article for color version of this figure.]
Figure 4.
Figure 4.
Overexpression of AOX reduces hydrogen peroxide and ROS accumulation in cassava roots. A, Roots were exposed to DAB and imaged using an Olympus DP20 light microscope. B, Roots were exposed to H2DCF-DA and imaged using a Zeiss LSM 510 laser confocal microscope. WT, Wild type.
Figure 5.
Figure 5.
Expression of Arabidopsis AOX reduces the onset of PPD. A, Fourteen days after harvest. B, Twenty-one days after harvest. C, PPD scores at 21 d were obtained using ImageJ image processing software based on the intensity of vascular discoloration. Roots with a PPD score below 4 were considered suitable for consumption and marketing. The error bars show sd. Statistical analysis was done by one-way ANOVA with Dunnett’s multiple comparison test. All transgenic lines were significantly different from the wild type (WT) at P ≤ 0.05.
Figure 6.
Figure 6.
Delayed PPD phenotype and reduced biomass was observed in some AOX transgenic lines from field trials. A, Root cross-sections were made every 2 cm. B, Three transgenic lines were used in the field trials, and analysis was done 5 and 10 d after harvest. C, Storage root weight was determined from wild-type (WT) and PAOX plants grown in the field in Puerto Rico for 12 months. E, Environment-exposed end of the root (n = 3 from three different plants of the same line). The error bars indicate se. Asterisks indicate statistical differences as determined by P < 0.005 in relation to the wild type.
Figure 7.
Figure 7.
The mechanism and control of PPD in cassava roots. Mechanical damage that occurs during harvesting operations initiates cyanogenesis by bringing linamarin and linamarase in contact. Cyanide (HCN) inhibits complex IV in the mitochondrial electron transfer chain. Inhibition of complex IV causes a burst of ROS production (shown as red bursts) at complexes I and III. This oxidative burst causes PPD. Overexpressing the mitochondrial AOX, which is insensitive to cyanide, prevents the overreduction of complexes I and III, thus lowering ROS production and delaying PPD. Reduction of ROS to control PPD can also be achieved by the overexpression of ROS scavengers (light blue dashed arrow). HNL, Hydroxynitrile lyase; SOD, superoxide dismutase.

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