iTRAQ‐based analysis of changes in the cassava root proteome reveals pathways associated with post‐harvest physiological deterioration

Abstract

SUMMARYThe short storage life of harvested cassava roots is an important constraint that limits the full potential of cassava as a commercial food crop in developing countries. We investigated the molecular changes during physiological deterioration of cassava root after harvesting using isobaric tags for relative and absolute quantification (iTRAQ) of proteins in soluble and non-soluble fractions prepared during a 96 h post-harvest time course. Combining bioinformatic approaches to reduce information redundancy for unsequenced or partially sequenced plant species, we established a comprehensive proteome map of the cassava root and identified quantitatively regulated proteins. Up-regulation of several key proteins confirmed that physiological deterioration of cassava root after harvesting is an active process, with 67 and 170 proteins, respectively, being up-regulated early and later after harvesting. This included regulated proteins that had not previously been associated with physiological deterioration after harvesting, such as linamarase, glutamic acid-rich protein, hydroxycinnamoyl transferase, glycine-rich RNA binding protein, b-1,3-glucanase, pectin methylesterase, maturase K, dehydroascorbate reductase, allene oxide cyclase, and proteins involved in signal pathways. To confirm the regulation of these proteins, activity assays were performed for selected enzymes. Together, our results show that physiological deterioration after harvesting is a highly regulated complex process involving proteins that are potential candidates for biotechnology approaches to reduce such deterioration.

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

“…Similarly, the level of glutathione dehydrogenase was significantly reduced 24 h after harvest. While the increase of APX abundance does not correlate with increased APX activities during early PPD progression (Owiti et al, 2011) (Supplemental Figure 3), our study also revealed that levels of enzymes involved in the reduction of ascorbate and dehydroascorbate are not significantly upregulated.…”

“…Previously, we used detergent to extend proteome analysis to the nonsoluble protein fraction of cassava roots (Owiti et al, 2011), which substantially increased proteome coverage. Here, we used SDS to extract total protein from cassava roots during PPD progression.…”

“…The number of proteins identified in those studies was limited by the method as well as the lack of a fully annotated cassava genome. To increase proteome coverage, we previously reported an iTRAQ-based matrix-assisted laser desorption/ionization time-of-flight approach using the Viridiplantae protein database to identify 1387 nonredundant protein groups (Owiti et al, 2011). With the recent release and annotation of the cassava genome (Prochnik et al, 2012), it is now possible to expand cassava proteome coverage and better characterize its modulation during PPD.…”

Procedures for evaluating the tolerance of cassava genotypes to postharvest physiological deterioration

“…Considered a complex enzymatic process, PPD (Owiti et al, 2011) begins 15 min after the root cutting, and becomes visible between 24 and 72 hours after harvest, rendering roots inappropriate for consumption (Sánchez et al, 2006;Reilly et al, 2007;Morante et al, 2010). The main symptoms of PPD include vascular streaking, which is a blue-black discoloration of the xylem parenchyma, followed by general discoloration of the parenchyma, forming vascular ridges (Reilly et al, 2003).…”

Discordance between protein and transcript levels detected by selected reaction monitoring

“…4 The iTRAQ approach, specifically, has been adapted to compare and quantify proteins in various plant species. [5][6][7][8][9][10] Digested peptides in each protein sample are labeled with different iTRAQ reagents and samples are then pooled. Therefore, samples can be analyzed by LC/MS at the same time.…”