Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis

Abstract

Background: Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage.

Results: The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development.

Conclusions: Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants.

Keywords: Apple fruit; Cell death mechanism; Chilling stress; Malus × domestica Borkh; Metabolomics; Senescence; Transcriptomics.

Fig. 1

Scald severity (1 = no scald, 2 = less than 25%, 3 = 25–50%, 4 = greater than 75% coverage) on ‘Granny Smith’ apple fruit stored in air at 1 °C for up to 183 days (6 months). Apples were treated immediately following harvest with 2000 μL L⁻¹ DPA or 1 mL L⁻¹ 1-MCP. Error bars represent standard error (adapted from metadata presented in [20])

Fig. 2

Multi-block Partial Least Squares Discriminate Analysis (MBPLS-DA) bi-plots of gene expression and metabolite level data from ‘Granny Smith’ apple peel from fruit stored in air at 1 °C for up to 183 days (6 months). Apples were treated immediately following harvest with 2000 μL L⁻¹ DPA or 1 mL L⁻¹ 1-MCP. PC1–2 (top) and PC1–3 (bottom) planes are represented. Shapes represent scores for each observation where symbol size increases with storage duration and symbol color represents scald severity. Emboldened text labels represent position of each response variable used for the model. Red points represent transcript and blue metabolite loadings where point size and opaqueness indicates each relative VIP score

Fig. 3

Pageman [92] over expression analysis of MBPLS-DA scald VIPs associated (scald; VIP_transpos) or not associated (healthy; VIP_transneg) with scald incidence. Red squares indicate relatively over expressed and blue under expressed categories within the scald-related VIP list. The Mdomestica_196 library and ORA_Fisher protocol were used form the analysis using Bonferroni correction and ORA cutoff = 1

Fig. 4

Superficial scald symptom severity (adapted from metadata presented in [20]), trimethyldodeca-2,7 (E), 9(E), 11-tetraen-6-ol (CTOL) levels and methanol levels in ‘Granny Smith’ apple peel sampled multiple times up to 183 days from air stored (0.5 °C) untreated (control) ‘Granny Smith’ apples. Red circles designate the time point at which increased production of CTOL or methanol began to increase. Error bars represent standard error

Fig. 5

Undirected, edge-weighted gene expression/metabolite correlation networks generated from the first 2 months data (top) and all 6 months (bottom). Correlations R² ≥ 0.700 were considered for network generation. Only nodes connected with 1 or more other nodes are included and, to highlight areas of connection, edges become more transparent as they diminish towards R² = 0.700. Circles and triangles represent metabolite and gene clusters, respectively. Node size indicates neighborhood connectivity; larger nodes are connected to more closely correlated neighbors. Node color indicates clustering coefficient with green = 0, yellow = 0.64, and red = 1. Letters designate regions of interest including first neighbors (R² ≥ 0.700) of CTOL—pre-symptomatic (a) or methanol-- symptomatic (d) networks with black outlined nodes, metabolites and transcripts linked with tissue that will develop healthy (b and e), and metabolites and transcripts with relatively elevated levels in unripe peel (c and f)

Fig. 6

Pageman [92] over expression analysis of MBPLS-DA scald VIPs and genes contained in the “refined” methanol or CTOL sub-networks associated (scald) or not associated (healthy) with scald incidence. Red squares indicate relatively over expressed and blue under expressed categories The Mdomestica_196 library and ORA_Fisher protocol were used form the analysis using Bonferroni correction and ORA cutoff = 1

Fig. 7

Methanol, methyl hexanoate, alcohol acyltransferase, and pectin methylesterase level in ‘Granny Smith’ apple peel from fruit stored in air at 1 °C for up to 183 days (6 months). Apples were treated immediately following harvest with 2000 μL L⁻¹ DPA or 1 mL L⁻¹ 1-MCP. Error bars represent standard error