Abstract
1-methylcyclopropene (1-MCP) in an ethylene receptor antagonist that blocks ethylene perception and downstream ripening responses in climacteric fruit imparting a longer shelf life. However, in European pear, the application of 1-MCP irreversibly obstructs the onset of system 2 ethylene production resulting in perpetually unripe fruit with undesirable quality. Application of exogenous ethylene, carbon dioxide and treatment to high temperatures is not able to reverse the blockage in ripening. We recently reported that during cold conditioning, activation of alternative oxidase (AOX) occurs pre-climacterically. In this study, we report that activation of AOX via exposure of 1-MCP treated ‘D’Anjou’ pear fruit to glyoxylic acid triggers an accelerated ripening response. Time course physiological analysis revealed that ripening is evident from decreased fruit firmness and increased internal ethylene. Transcriptomic and functional enrichment analyses revealed genes and ontologies implicated in glyoxylic acid-mediated ripening, including AOX, TCA cycle, fatty acid metabolism, amino acid metabolism, organic acid metabolism, and ethylene-responsive pathways. These observations implicate the glyoxylate cycle as a biochemical hub linking multiple metabolic pathways to stimulate ripening through an alternate mechanism. The results provide information regarding how blockage caused by 1-MCP may be circumvented at the metabolic level, thus opening avenues for consistent ripening in pear and possibly other fruit.
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Introduction
Every year, 1.6 billion tons of food goes to waste. This is about one-third of the food that is produced for human consumption1. Unpredictable ripening of fruit is one of the main causes of loss after harvest. This is particularly true of climacteric fruits, which are characterized by a spike in ethylene biosynthesis, known as system 2 (S2) ethylene production, and a concomitant burst in respiration at the onset of ripening. The ethylene receptor antagonist 1-methylcyclopropene (1-MCP) is used to impart a longer shelf life by limiting the ethylene perception and activation of downstream ripening responses2,3,4.
Uniquely, in European pear fruit (Pyrus communis), 1-MCP treatment may irreversibly inhibit endogenous or system 2 ethylene production and the respiratory climacteric5,6. Furthermore, exogenous ethylene application does little to affect the capacity of 1-MCP-treated pears to ripen7,59.
Functional annotation
The master transcriptome fasta produced from the Illumina assembly was imported into OmicsBox 1.1.135 (BioBam Bioinformatics S.L., Valencia, Spain) for functional annotation of expressed contigs. Contig sequences were identified by a blastx alignment against the NCBI ‘Viridiplantae’ database with and e-value specification of 10.0E-3. GO annotation was assigned using the ‘Map**’ and ‘Annotation’ features using default parameters to generate a functionally annotated master assembly60.
Differential expression analysis
Temporally differentially expressed genes were identified using the time course, multi-series differential expression feature in the OmicsBox suite, which employs the maSigPro R package61. The FDR cutoff value was set to 0.05. The statistical analysis ensured that genes that did not meet the assumption of equal variances were eliminated from the analysis, which was particularly important given that the three experiments were performed at different times throughout the 2018 season. The DEGs and expression values were matched with their corresponding functional annotations (Supplementary File 5).
GO enrichment analysis
Gene ontology (GO) enrichment analysis was conducted to determine over and underrepresented biological processes, molecular functions, and cellular components among the differentially expressed sequences using the OmicsBox Enrichment Analysis (Fisher’s Exact Test) function60 (Supplementary File 8). The annotated master transcriptome was used as the reference dataset, and the set of genes identified as differentially expressed over time in the treatment group versus the control group was used as the test dataset.
qRT-PCR validation
Primers for qRT-PCR targeting seven differentially expressed genes in the ripening-related pathways discussed previously were designed using the NCBI Primer-BLAST tool62. 200 ng RNA for each sample was used to generate 1st strand cDNA using the Invitrogen VILO kit (Life Technologies, Carlsbad, CA USA). cDNA preparations were then diluted to 20 ng/uL. Final library concentrations were quantified using a Qubit fluorometer (Carlsbad, CA). qRT-PCR technical replicate reactions were prepared for each of the gene targets using the iTAQ Universal SYBR Green Supermix with ROX reference dye (BioRad, Hercules, CA) per the manufacturer’s protocols with 20 ng of template cDNA. In a Stratagene MX3005P, the following thermocycler profile was used: 95 °C initial disassociation for 2:30 minutes followed by 50 amplification cycles (95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s) and a final, single-cycle phase to generate a dissociation curve (95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s). The LinRegPCR tool was used to calculate the Cq values for each reaction63,64 (Supplementary File 10). Cq values, which were calculated from efficiency scores below 1.80 or above 2.20 were considered sufficiently low in confidence and were deemed unacceptable and were omitted from the analysis.
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Acknowledgements
The authors thank Blue Star Growers (Cashmere, WA) for their support and for providing pears for conditioning experiments, Scott Mattinson for assistance with the gas chromatography work, and Coleman Schnebly for assisting with measurement of physiological parameters. Work in the Dhingra lab was supported in part by Washington State University Agriculture Center Research Hatch Grant WNP00011 and grant funding from Fresh and Processed Pear Research Subcommittee to AD. SLH acknowledges the support received from ARCS Seattle Chapter and National Institutes of Health/National Institute of General Medical Sciences through an institutional training grant award T32-GM008336. The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or NIH.
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S.L.H. and A.D. designed the study. S.L.H. performed the experiments. R.G. performed the HPLC analysis S.L.H. and A.D. performed the data analysis. All authors prepared, edited, and approved the manuscript.
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Hewitt, S.L., Ghogare, R. & Dhingra, A. Glyoxylic acid overcomes 1-MCP-induced blockage of fruit ripening in Pyrus communis L. var. ‘D’Anjou’. Sci Rep 10, 7084 (2020). https://doi.org/10.1038/s41598-020-63642-z
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DOI: https://doi.org/10.1038/s41598-020-63642-z
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