Evidence for the Involvement of Vernalization-related Genes in the Regulation of Cold-induced Ripening in ‘D’Anjou’ and ‘Bartlett’ Pear Fruit

Abstract

European pear (Pyrus communis L.) cultivars require a genetically pre-determined duration of cold-temperature exposure to induce autocatalytic system 2 ethylene biosynthesis and subsequent fruit ripening. The physiological responses of pear to cold-temperature-induced ripening have been well characterized, but the molecular mechanisms underlying this phenomenon continue to be elucidated. This study employed established cold temperature conditioning treatments for ripening of two pear cultivars, 'D'Anjou' and 'Bartlett'. Using a time-course transcriptomics approach, global gene expression responses of each cultivar were assessed at four different developmental stages during the cold conditioning process. Differential expression, functional annotation, and gene ontology enrichment analyses were performed. Interestingly, evidence for the involvement of cold-induced, vernalization-related genes and repressors of endodormancy release was found. These genes have not previously been described to play a role in fruit during the ripening transition. The resulting data provide insight into cultivar-specific mechanisms of cold-induced transcriptional regulation of ripening in European pear, as well as a unique comparative analysis of the two cultivars with very different cold conditioning requirements.For both cultivars, fruit softening accelerated once the fruit was transferred to 20°C. The rate of softening was more rapid for 'Bartlett' than 'D'Anjou'. RNAseq Assembly AnalysisRNAseq assembly generated 140077 contigs (Supplementary File 1). In the OmicsBox suite, the maSigPro R package was used to conduct time course differential expression analyses for both cultivars. 17,711 differentially expressed contigs (p<0.05) were identified for 'D'Anjou', with 7,174 of these contigs exhibiting significant linear or quadratic trends over time (R>0.8). In 'Bartlett' 31,481 contigs were identified as being differentially expressed, with 7,174 contigs exhibiting significant quadratic or linear trends over time (R>0.8) (Supplementary File 2). Similarities and differences in expression trends of contigs of interest between 'D'Anjou' and 'Bartlett', as well as expression patterns of differentially expressed contigs (DECs) associated with ethylene and phytohormone metabolism, abscisic acid metabolism, TCA cycle, respiration, were assessed. Additionally, in order to better understand the mechanisms underlying the chilling requirement for ripening in Pyrus, the expression of genes associated with: vernalization, flowering, dormancy, and other processes directly induced by cold/chilling were observed. Preclimacteric expression of AOX1 peaked during conditioning prior to onset of ripening (Figure 1), supporting the hypothesis. Additional key genes, including those that mediate vernalization and endodormancy release were also observed to be differentially expressed. Detailed analysis of RNAseq results and enriched gene ontologies related to phytohormone metabolism and coldresponse pathways are discussed in detail in the following sections.

Beyond Ethylene: New Insights Regarding the Role of AOX in the Respiratory Climacteric

“…Recent studies provide new insights into the role of AOX in fruit ripening, and how exogenous stimulation (e.g. chemical or temperature) can be utilized to intentionally modulate ripening responses [24,30,56,57]. Results of physiology and gene expression studies of cold conditioned pear fruit suggest that pre-climacteric activity of AOX facilitating the S1-S2 transition directly impacts the ripening process in these fruit [24,30,57].…”

“…chemical or temperature) can be utilized to intentionally modulate ripening responses [24,30,56,57]. Results of physiology and gene expression studies of cold conditioned pear fruit suggest that pre-climacteric activity of AOX facilitating the S1-S2 transition directly impacts the ripening process in these fruit [24,30,57]. Furthermore, exogenous chemical manipulation of AOX activity with hydrogen sulfide and glyoxylic acid to modulate ripening in pre-climacteric fruit has also been demonstrated [56,58].…”

“…Furthermore, exogenous chemical manipulation of AOX activity with hydrogen sulfide and glyoxylic acid to modulate ripening in pre-climacteric fruit has also been demonstrated [56,58]. Transcriptomic characterization of expressed genes in response to cold conditioning and to glyoxylic acid application in pear fruit has provided insight into novel ripening-associated pathways and support the putative roles of the AOX alternative respiratory pathway and the glyoxylate cycle in the larger ripening network [24,58].…”

“…Not surprisingly, manipulation of environmental factors, genetic factors, and use of chemical inhibitors like 1-methylcyclopropene (1-MCP) to inhibit ripening result in developmental patterns that don't follow the classical model of ethylene response and signaling [19][20][21][22]. Mechanisms for blockage and/or bypass of the concerted steps in classical ethylene biosynthesis are beginning to be elucidated as more studies examine variations in the classical paradigm of ripening via genetic manipulation or exogenous perturbation by temperature or chemicals [23,24]. As molecular biology, transcriptomics, and epigenetic analysis tools have rapidly advanced, new insights have been gained into some of the master regulators of ripening acting upstream of ethylene [25,26].…”

Glyoxylic acid overcomes 1-MCP-induced blockage of fruit ripening in Pyrus communis L. var. ‘D’Anjou’

“…Interestingly, 'response to cold', 'cellular oxidant detoxification' and 'antioxidant activity' were also enriched. While these experiments were conducted at ambient temperature, cold conditioning is required for pear fruit to ripen [5], and recent studies of cold-induced ripening of pear implicate the involvement of AOX for crosstalk with ethylene and scavenging of reactive oxygen species (ROS), thereby alleviating oxidative damage under external stress conditions [15,47].…”