Abstract
Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by proteomic analysis.
Highlights
A The total number of protein IDs indicates the total protein IDs identified by two sets of biological replicate samples. b The number of unique proteins from each sets denotes the number of protein IDs exclusively identified from each of the two sets
The immunoblot analysis showed that serine hydroxymethyltransferase (SHMT) (Locus_304_Contig7) protein levels were up-regulated in plantain following cold stress and partially returned to normal during the 24 h recovery, but no substantial change was induced by the cold treatment in banana. -Hex (Locus_1367_Contig4) showed a diametrically opposite response to cold stress in plantain and banana: levels steadily decreased in plantain at C6h and C24h, but slowly increased in banana over the time period of the cold stress and recovery point
Research Challenges for Plantain and Banana as Nonmodel Organisms—Banana and plantain are grown in more than one hundred tropical and subtropical countries and collectively are the fourth most consumed staple food worldwide (17). Despite their major societal and economic importance, the lack of fully sequenced genome for banana and plantains has limited the application of the high-throughput “omics” technologies
Summary
Chemicals and Materials—Sequence-grade acetonitrile (ACN), trifluoroacetic acid (TFA), and formic acid (FA) were purchased from Fisher Scientific (Fair Lawn, NJ). Protein Extraction, Digestion, and iTRAQ Labeling—Plantain leaf proteins from two biological replicate samples were obtained by grinding tissues in liquid N2 followed by suspension in ice-cold sodium phosphate buffer (100 mM, pH 7.5) containing 1 mM EDTA, 100 g/ml PMSF, and 0.1% Triton X-100. For lipoxygenase activity assays (43), 0.5 g of lyophilized, powdered plantain or banana leaves was homogenized in 1 ml of extraction solution, The resulting homogenates were centrifuged at 12,000 ϫ g for 20 min at 4 °C and the supernatant was recovered and stored on ice. Protein concentrations were determined by the Bradford assay (35) with bovine serum albumin as the standard. One activity unit (U) was defined as the increase in one unit of absorbance at 234 nm minϪ1, and the results were expressed as specific activity (U mg proteinϪ1)
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