Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots

Abstract

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is the result of a rapid oxidative burst, which leads to discoloration of the vascular tissues due to the oxidation of phenolic compounds. In this study, coexpression of the reactive oxygen species (ROS)-scavenging enzymes copper/zinc superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) in transgenic cassava was used to explore the intrinsic relationship between ROS scavenging and PPD occurrence. Transgenic cassava plants integrated with the expression cassette p54::MeCu/ZnSOD-35S::MeCAT1 were confirmed by Southern-blot analysis. The expression of MeCu/ZnSOD and MeCAT1 was verified by quantitative reverse transcription-polymerase chain reaction and enzymatic activity analysis both in the leaves and storage roots. Under exposure to the ROS-generating reagent methyl viologen or to hydrogen peroxide (H2O2), the transgenic plants showed higher enzymatic activities of SOD and CAT than the wild-type plants. Levels of malondialdehyde, chlorophyll degradation, lipid peroxidation, and H2O2 accumulation were dramatically reduced in the transgenic lines compared with the wild type. After harvest, the storage roots of transgenic cassava lines show a delay in their PPD response of at least 10 d, accompanied by less mitochondrial oxidation and H2O2 accumulation, compared with those of the wild type. We hypothesize that this is due to the combined ectopic expression of Cu/ZnSOD and CAT leading to an improved synergistic ROS-scavenging capacity of the roots. Our study not only sheds light on the mechanism of the PPD process but also develops an effective approach for delaying the occurrence of PPD in cassava.

Figure 1.

Analyses of cassava SOD and CAT transcript and abundance and phenotypic evaluation of transgenic plants harvested from the field. A, qRT-PCR analysis of MeCu/ZnSOD and MeCAT1 expression levels both in wild-type (WT) and transgenic cassava. Total RNA was extracted from leaves, and the data are shown relative to the wild type, using β-actin as an internal control. Data are presented as means ± sd of three independent RNA samples. B, SOD and CAT isoforms in leaves of wild-type and transgenic plants detected by activity staining of a nondenaturing polyacrylamide gel. Three SOD isoforms, MnSOD, FeSOD, and Cu/ZnSOD, are indicated. A substantial increase of the cytoplasmic Cu/ZnSOD is highlighted by the black arrowhead. The Rubisco LSU protein was used as a loading control. C and D, Normal growth of transgenic plants with fully developed storage roots (C) and unchanged yield (D) in comparison with the wild type in the field. Bars = 15 cm. No significant difference was found by Duncan’s multiple comparison tests at P < 0.05. [See online article for color version of this figure.]

Figure 2.

Changes in protoplast viability and mitochondrial membrane integrity in the presence of 1 m H₂O₂. A, The viability of cassava mesophyll protoplasts after H₂O₂ treatment was estimated by fluorescein diacetate staining. Data are represented as means of six replicates ± sd (more than 300 cells were counted for each experiment per genotype). Values labeled with different letters (a, b, and c) are significantly different by Duncan’s multiple comparison tests at P < 0.05. B, Mitochondrial membrane integrity under 1 m H₂O₂ stress. Cassava protoplasts were stained with rhodamine 123, and the fluorescent signal was observed with a confocal microscope. Bars = 5 μm. [See online article for color version of this figure.]

Figure 3.

Enhanced tolerance to H₂O₂-mediated oxidative stress in transgenic leaves. A, H₂O₂ accumulation in leaves detected by DAB staining. Bars = 0.5 cm. B, Changes in the levels of H₂O₂ concentration between wild-type (WT) and transgenic cassava during 0.5 m H₂O₂ treatment. C and D, Changes in SOD (C) and CAT (D) activities between wild-type and transgenic cassava during H₂O₂ treatment. Values represent means of three independent experiments ± sd. Values labeled with different letters (a, b, and c) are significantly different by Duncan’s multiple comparison tests at P < 0.05. [See online article for color version of this figure.]

Figure 4.

Enhanced tolerance to MV-mediated oxidative stress in transgenic leaves. A, Leaves treated with 100 µm MV showing the senescence phenotype of wild type (WT) plants and the stay-green phenotype of transgenic plants. Bar = 0.5 cm. B and C, Chlorophyll and MDA contents in the first leaf of MV-treated and untreated plants. FW, Fresh weight. D and E, Changes in SOD and CAT activities between wild-type and transgenic cassava during MV treatment. Data are presented as means ± sd from triplicate independent measurements. Values labeled with different letters (a, b, and c) at the same time point are significantly different by Duncan’s multiple comparison tests at P < 0.05. [See online article for color version of this figure.]

Figure 5.

Delayed PPD occurrence of storage roots from transgenic cassava by visual and fluorescence determination. A, Visual detection of PPD occurrence using the International Center for Tropical Agriculture method (Wheatley et al., 1985; Morante et al., 2010). The levels of vascular discoloration are indicated by percentages using ImageJ processing software. B, Inhibition of mitochondrial ROS generation during the PPD process in transgenic storage roots detected by fluorescent probes. The fluorescence intensity of oxidized rhodamine was observed with a fluorescence microscope (Zeiss LSM 510 META) with excitation/emission of 488/515 nm and 635/680 nm for DHR and MitoTracker-Deep Red FM, respectively. a, Xylem vessel; b, bundle sheath. Bars = 20 μm. C, Changes of H₂O₂ concentrations during PPD between wild-type (WT) and transgenic cassava. Values labeled with different letters (a, b, and c) at the same time point are significantly different by Duncan’s multiple comparison tests at P < 0.05. FW, Fresh weight. [See online article for color version of this figure.]

Figure 6.

Expression and activity patterns of SOD and CAT enzymes during the PPD process. A and B, Overall up-regulated expression of MeCu/ZnSOD and MeCAT1 during PPD in transgenic plants compared with the wild type (WT). C and D, Higher levels of SOD and CAT activities during PPD in transgenic cassava compared with the wild type. Values labeled with different letters (a, b, and c) at the same time point are significantly different by Duncan’s multiple comparison tests at P < 0.05.

Figure 7.

Schematic illustration of the intrinsic relationship between ROS production, ROS scavenging, and ROS homeostasis for regulating PPD in cassava storage roots. Exposure to oxygen or mechanical wounding during harvest leads to increased ROS production in the storage root. Inefficient endogenous ROS scavenging results in excess ROS, which triggers rapid PPD responses, and stable ROS homeostasis in harvested cassava roots is never achieved. Ectopic expression of SOD and CAT leads to timely scavenging of excess ROS, thereby keeping the ROS homeostasis balanced and delaying PPD occurrence. [See online article for color version of this figure.]