Large-Scale Proteomics of the Cassava Storage Root and Identification of a Target Gene to Reduce Postharvest Deterioration

Abstract

Cassava (Manihot esculenta) is the most important root crop in the tropics, but rapid postharvest physiological deterioration (PPD) of the root is a major constraint to commercial cassava production. We established a reliable method for image-based PPD symptom quantification and used label-free quantitative proteomics to generate an extensive cassava root and PPD proteome. Over 2600 unique proteins were identified in the cassava root, and nearly 300 proteins showed significant abundance regulation during PPD. We identified protein abundance modulation in pathways associated with oxidative stress, phenylpropanoid biosynthesis (including scopoletin), the glutathione cycle, fatty acid α-oxidation, folate transformation, and the sulfate reduction II pathway. Increasing protein abundances and enzymatic activities of glutathione-associated enzymes, including glutathione reductases, glutaredoxins, and glutathione S-transferases, indicated a key role for ascorbate/glutathione cycles. Based on combined proteomics data, enzymatic activities, and lipid peroxidation assays, we identified glutathione peroxidase as a candidate for reducing PPD. Transgenic cassava overexpressing a cytosolic glutathione peroxidase in storage roots showed delayed PPD and reduced lipid peroxidation as well as decreased H₂O₂ accumulation. Quantitative proteomics data from ethene and phenylpropanoid pathways indicate additional gene candidates to further delay PPD. Cassava root proteomics data are available at www.pep2pro.ethz.ch for easy access and comparison with other proteomics data.

Figure 1.

Experimental and Bioinformatics Workflow of the Proteome Analysis. Total proteins were extracted from roots, separated by gel electrophoresis, digested with trypsin, and analyzed by liquid chromatography–MS/MS using an LTQ-Orbitrap device. The workflow references Supplemental Data Sets 1 to 5, corresponding to the analysis steps.

Figure 2.

Molecular Function Categories Overrepresented in the Proteins Regulated during PPD. Category overrepresentation was performed with Arabidopsis identifiers and determined relative to the background set of all identified proteins for which an Arabidopsis identifier was available to take into account the bias introduced by the extraction and detection methods.

Figure 3.

Detection and Regulation of Proteins Involved in the Ascorbate/Glutathione Cycle. Regulated proteins are selected based on the Progenesis ANOVA analysis (P < 0.05), and regulation is represented graphically using normalized protein abundance and sd (n = 3).

Figure 4.

Enzymatic Activities in Protein Fractions from Collected Time Points. Protein fractions were GPX (A), GR (B), and GST (C). Means ± sd of three biological replicates are shown (Student’s t test, *P < 0.05).

Figure 5.

PPD Score Analysis of Wild-Type and Transgenic Cassava Storage Roots. (A) Comparison of the PPD scores of the control line and transgenic PAT-GPX lines at 6, 12, 24, and 48 h after harvest (Student’s t test, *P < 0.05). (B) Root slice of the control line at 24 h after harvest. (C) Root slice of the PAT-GPX2 line at 24 h after harvest. [See online article for color version of this figure.]