Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene

Abstract

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5-20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.

Keywords: Citrus limon; 1-methylcyclopropene; carotenoids; chlorophyll; ethylene; low temperature; peel degreening; phytohormones; transcriptome.

Fig. 1.

Ethylene-induced peel degreening in detached lemon fruit. (A) Appearance and colour index of fruit in response to treatment with ethylene and/or 1-methylcyclopropene (1-MCP). Fruit were either untreated (control), continuously treated with 100 µl l^–1 ethylene (ET), treated with 2 µl l^–1 1-MCP for 12 h (1-MCP), or pre-treated with 2 µl l^–1 1-MCP for 12 h followed by continuous treatment with 100 µl l^–1 ethylene (1-MCP+ET). An increase in the colour index indicates the fruit changing from green to yellow. (B) Changes in peel chlorophyll contents in response to ethylene and/or 1-MCP treatments. (C) Changes in peel carotenoid contents in response to ethylene and/or 1-MCP treatments. Data are means (±SE) of five replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 2.

Promotion of peel degreening by low temperature in detached lemon fruit. (A) Appearance and colour index of fruit during storage at 5–25 °C in an ethylene-free environment. An increase in the colour index indicates the fruit changing from green to yellow. (B) Effects of 1-MCP treatment on peel colour changes during storage at 5, 15, or 25 °C. Treatments with 1-MCP (2 µl l^–1) were carried out twice a week to keep fruit insensitive to ethylene. (C) Peel chlorophyll contents at different storage temperatures with or without 1-MCP treatment. (D) Peel carotenoid contents during storage at different temperatures with or without 1-MCP treatment. Data are means (±SE) of five replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05)

Fig. 3.

Global transcriptome changes induced by ethylene treatment and low temperature treatments in the flavedo of detached lemon fruit. (A) The numbers of differentially expressed genes (DEGs) in response to ethylene (ET), 5 °C, and 15 °C. (B) Venn diagrams showing the up- and down-regulated DEGs in response to ethylene, 5 °C, and 15 °C. (C) Weighted gene co-expression network analysis (WGCNA) and subsequent heatmap visualization of the 3105 DEGs from RNA-seq data. Each end branch in the tree represents one of the DEGs and each major branch represents one of the modules. The panel with total gene numbers shows the eight different modules obtained after implementing the tree cut-off (0.15) to merge close modules. The panel at the right shows a heatmap visualization of the expression measures of DEGs in each module in response to ethylene and the different storage temperatures. (D) Selected GO terms enriched among the WGCNA co-expressed DEGs in response to ethylene, 5 °C, and 15 °C.

Fig. 4.

Expression of genes related to chlorophyll metabolism in the flavedo of detached lemon fruit in response to ethylene or low temperatures. (A) Heatmap showing identified differentially expressed genes associated with chlorophyll metabolism in fruit exposed to ethylene, 5 °C, or 15 °C. The data are expressed as the log₂ value of fold-change for ethylene (ET) versus control, 5 °C versus 25 °C, and 15 °C versus 25 °C. (B) RT-qPCR analysis of the expression of chlorophyllase 1 (ClCLH1) and pheophytinase (ClPPH), indicated by the arrows in (A), which are known to be involved in chlorophyll degradation in many plant species. Expression is relative to the value at harvest and the housekeeping gene was AcActin. Data are means (±SE) of three replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 5.

Expression of genes related to carotenoid metabolism in the flavedo of detached lemon fruit in response to ethylene or low temperatures. (A) Heatmap of identified differentially expressed genes associated with carotenoid metabolism in fruit exposed to ethylene, 5 °C, or 15 °C. The data are expressed as the log₂ value of fold-change for ethylene (ET) versus control, 5 °C versus 25 °C, and 15 °C versus 25 °C. (B) RT-qPCR analysis of the expression of phytoene synthase 1 (ClPSY1), lycopene cyclase 2a (ClLCYb2a), and β-carotene hydroxylase 1 (ClCHYb1), indicated by the arrows in (A). Expression is relative to the value at harvest and the housekeeping gene was AcActin. Data are means (±SE) of three replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 6.

Changes in the expression of genes encoding photosystem proteins in the flavedo of detached lemon fruit in response to ethylene or low temperatures. (A) Heatmap of identified differentially expressed genes encoding photosystem proteins in fruit exposed to ethylene, 5 °C, or 15 °C. The data are expressed as the log₂ value of fold-change for ethylene (ET) versus control, 5 °C versus 25 °C, and 15 °C versus 25 °C. (B) RT-qPCR analysis of the expression of light harvesting complex 2 (ClLHCB2), indicated by the arrow in (A). Expression is relative to the value at harvest and the housekeeping gene was AcActin. Data are means (±SE) of three replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 7.

Levels of phytohormones and the expression of associated genes in the flavedo of detached lemon fruit. (A) Heatmap showing differentially expressed genes encoding proteins associated with phytohormone biosynthesis and signalling in fruit exposed to ethylene or low temperatures. The data are expressed as the log₂ value of fold-change for ethylene (ET) versus control, 5 °C versus 25 °C, and 15 °C versus 25 °C. (B) RT-qPCR analysis of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 5 (ClNCED5), indicated by the arrow in (A). Expression is relative to the value at harvest and the housekeeping gene was AcActin. (C) Levels of ABA and JA-Ile in lemon fruit treated with ethylene or after 28 d storage at the indicated temperatures. Data are means (±SE) of three replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 8.

Changes in expression of genes encoding transcription factors in the flavedo of detached lemon fruit. (A) Heatmap showing identified differentially expressed genes encoding various transcription factors in fruit exposed to ethylene or low temperatures. The data are expressed as the log₂ value of fold-change for ethylene (ET) versus control, 5 °C versus 25 °C, and 15 °C versus 25 °C. (B–D) RT-qPCR analysis of the relative expression of (B) ClERF114, (C) ClERF3, and (D) ClbHLH25, as indicated by the arrows in (A). Expression is relative to the value at harvest and the housekeeping gene was AcActin. Data are means (±SE) of three replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 9.

Changes in peel colour and gene expression analysis in lemon fruit during on-tree maturation. (A) Appearance and citrus colour index of representative fruit at different developmental stages together with data for minimum ambient temperatures. An increase in the colour index indicates the fruit changing from green to yellow. The temperature data 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). (B) Chlorophyll a and b contents at different developmental stages. (C) Levels of lutein, α-carotene, and β-carotene at different developmental stages. (D–F) RT-qPCR analysis of the expression of selected genes associated with chlorophyll metabolism and photosystem proteins (D), carotenoid metabolism (E), and transcription factors (F) at different developmental stages. Expression is relative to the value at harvest and the housekeeping gene was AcActin. Data are means (±SE) of five replicate fruits. Different letters indicate significant differences between means as determined using ANOVA followed by Tukey’s test (P<0.05).

Fig. 10.

Proposed model for the distinct ethylene-dependent and low temperature-modulated peel degreening in lemon fruit. Uniquely ethylene-regulated transcription factors (TFs) are represented by ClERF114 while low temperature-specific TFs are represented by ClERF3. An additional set of TFs (represented by ClbHLH25) are shared between the ethylene and low-temperature regulatory pathways. Exposure to ethylene or low temperature induces changes in the levels of these unique and shared TFs, triggering the expression of various genes associated with chlorophyll degradation (green), carotenoid metabolism (orange), photosystem proteins (blues), and phytohormones (red).