Examining the Role of Low Temperature in Satsuma Mandarin Fruit Peel Degreening via Comparative Physiological and Transcriptomic Analysis

Abstract

Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4-6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10°C and 15°C after 28-42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5°C, 20°C, and 25°C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulated by (i) propylene (and by analogy, ethylene) alone, (ii) low temperature (5°C, 10°C, or 15°C vs. 25°C) alone, and (iii) either propylene or low temperature. Gene-encoding proteins associated with chlorophyll degradation (such as CuSGR1, CuNOL, CuACD2, CuCAB2, and CuLHCB2) and a transcription factor (CuERF114) were differentially expressed by propylene or low temperature. To further examine temperature-induced pathways, we also monitored gene expression during on-tree fruit maturation vs. postharvest. The onset of on-tree peel degreening coincided with autumnal drops in field temperatures, and it was accompanied by differential expression of low temperature-regulated genes. On the contrary, genes that were exclusively regulated by propylene (such as CuCOPT1 and CuPOX-A2) displayed insignificant expression changes during on-tree peel degreening. These findings indicate that low temperatures could be involved in the fruit ripening-related peel degreening independently of ethylene.

Keywords: RNA-Seq; chlorophyll; citrus; degreening; ethylene; on-tree; storage.

Figure 1

Characterization of ethylene-induced peel degreening in postharvest Satsuma mandarin fruit. (A) Appearance of fruit during continuous treatment with propylene, an analog of ethylene. (B) Changes in citrus color index. Dashed line indicates color index 0, which represents a color change from green to yellow/orange. (C) Changes in peel chlorophyll content. Propylene treatment was carried out continuously at 25°C to induce ethylene signaling. Each data point represents the mean (±SE) of six replicate fruit. Different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05).

Figure 2

Peel degreening behavior of Satsuma mandarin fruit at different storage temperatures. (A) Appearance of fruit during and after storage at different temperatures. (B) Changes in citrus color index during and after storage at different temperatures. Dashed line indicates color index 0, which represents a color change from green to yellow/orange. (C) Changes in peel chlorophyll content during storage at different storage temperatures. Samples for chlorophyll content determination were collected in 2017. Each data point represents the mean (±SE) of six replicate fruit. Different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05).

Figure 3

Peel color changes in Satsuma mandarin fruit during on-tree maturation. (A) Appearance of fruit during on-tree maturation. (B) Changes in citrus color index and minimum field temperatures. Data for temperature were accessed from the website of Japan Meteorological Agency (http://www.data.jma.go.jp/obd/stats/etrn/view/daily_s1.php?prec_no=72&block_no=47891&year=2014&month=12&day=&view=p1). (C) Changes in the peel content of chlorophyll a and chlorophyll b. Data points represent the mean (±SE) of five fruit and different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05). Samples for on-tree assessments were collected in 2017. DAFB, days after full bloom.

Figure 4

Transcriptomic changes in the flavedo of Satsuma mandarin fruit in response to propylene and different storage temperatures. (A) Number of genes that were differentially expressed in response to propylene treatment and storage at 5°C, 10°C, 15°C, and 20°C. Propylene effect was determined using at-harvest (0 days) samples as a control, whereas the control samples for storage temperature tests included fruit at 25°C. (B) Weighted gene coexpression network analysis (WGCNA) of differentially expressed genes (DEGs) identified from the flavedo of Satsuma mandarin fruit treated with propylene or stored at different temperatures. The left panel shows the cluster dendrogram with the major tree branches. The right panel shows the different colors assigned to each one of the 17 modules obtained after implementing the tree cut line (0.15) to merge close modules and the number of genes in each module. (C) Heatmap showing the expression measures of genes in each WCGNA module at harvest (0 days), after 4 days propylene treatment and 28 days storage at the specified temperatures. (D) Selected gene ontology (GO) terms that were enriched among the DEGs that responded to propylene treatment or storage at 5°C, 10°C, and 15°C. Color panels display the P-values of the respective GO enrichment terms.

Figure 5

RT-qPCR analysis of the expression of genes associated with chlorophyll degradation and a transcription factor selected from the RNA-Seq data. (A) CuSGR1 (Ciclev10021651m.g), (B) CuNOL (Ciclev10008039m.g), (C) CuACD2 (Ciclev10026248m.g), (D) CuCAB2 (Ciclev10016286m.g), (E) CuLHCB2 (Ciclev10016280m.g), and (F) CuERF114 (Ciclev10032575m.g). Data points represent the mean (±SE) of three fruit and different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05).

Figure 6

Relative expression levels of selected genes associated with chlorophyll degradation and a transcription factor in the flavedo of Satsuma mandarin fruit during on-tree peel degreening. Values below the horizontal axis indicate the average minimum environmental temperature recorded on the specified date. (A) CuSGR1 (Ciclev10021651m.g), (B) CuNOL (Ciclev10008039m.g), (C) CuACD2 (Ciclev10026248m.g), (D) CuCAB2 (Ciclev10016286m.g), (E) CuLHCB2 (Ciclev10016280m.g), and (F) CuERF114 (Ciclev10032575m.g). Data points represent the mean (±SE) of three fruit and different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05). Samples for on-tree assessments were collected in 2017. DAFB, days after full bloom.

Figure 7

Relative expression levels of ethylene-specific (A,B) and low temperature-specific (C) genes during postharvest and on-tree peel degreening in Satsuma mandarin fruit. Values below the horizontal axis of on-tree graphs indicate the days after full bloom. CuCOPT1 (Ciclev10030036m.g), CuPOX-A2 (Ciclev10015790m.g), and CuERF3 (Ciclev10009593m.g). Data points represent the mean (±SE) of three fruit and different letters indicate significant differences in ANOVA (Tukey's test, P < 0.05).