Effects of Postharvest Treatment with 1-Naphthaleneacetic Acid on Chlorophyll and Carotenoid Metabolism in Citrus Fruit

Abstract

‘Miyagawa-wase’ (Citrus unshiu Marcow.), an early-season citrus variety, is rich in multiple nutrients and widely consumed in Japan. In ‘Miyagawa-wase’, when the pulp reaches maturity and is ready to eat, the peel is still a greenish color because of the relatively high temperature in the harvest season. In this study, to improve the coloration of ‘Miyagawa-wase’ peel, we treated the fruit with 1-naphthaleneacetic acid (NAA) after harvest. The results showed that postharvest treatment with NAA was effective to induce chlorophyll degradation and carotenoid accumulation in ‘Miyagawa-wase’ peel during storage. In the NAA treatment, the reduction in the chlorophyll contents after harvest was in parallel with decreases in the expression of chlorophyll biosynthetic genes (CitGGDR, CitCHLH, CitCHL27, CitPORA, and CitCAO) and an increase in chlorophyll degradation gene CitPPH. The contents of the major carotenoids, β-cryptoxanthin and 9-cis-violaxanthin, were increased by the NAA treatment through upregulation of the expression of carotenoid biosynthetic genes (CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb) after harvest. In addition, it was found that the positive effect on degreening in the NAA treatment was inhibited by the ethylene antagonist 1-MCP. In the combination treatment using NAA and 1-MCP, the total chlorophyll content was much higher, while the contents of β-cryptoxanthin and 9-cis-violaxanthin were lower compared with NAA treatment alone, indicating that the acceleration of degreening by NAA may be caused by ethylene. The results presented in this study suggest that postharvest NAA treatment is an effective method for improving the peel coloration in early-season citrus varieties.

Introduction

In citrus fruit, peel color turns from green to orange gradually during the ripening process. The color development in citrus fruit is closely associated with the seasonal temperature. It is well known that low temperature is a key factor to stimulate color break in citrus fruit peel (Alquézar et al., 2008; Rodrigo et al., 2013), while high temperature inhibits peel color development during the ripening process. In general, the color development in citrus fruit requires a night time temperature below 13°C and a large difference in temperature between day and night (Mitalo et al., 2020). However, in some early-season citrus varieties, the temperature does not drop low enough for peel color development in the harvest season, and the peel is often a greenish color when the pulp reaches maturity. The peel color is the main index of fruit quality, and greenish peel color affects the acceptance of citrus fruit by consumers. Thus, postharvest degreening treatment is of great importance for early-season citrus varieties to improve coloration and gain more acceptance for marketing.

In most citrus varieties, chlorophyll and carotenoid are the major pigments determining the fruit color. During the ripening process, the rapid loss of chlorophylls and massive accumulation of carotenoids lead to fruit degreening (Rodrigo et al., 2013). In the past decades, the metabolism of chlorophyll and carotenoids have been extensively investigated in citrus fruit, and the key enzymes involved in chlorophyll and carotenoid accumulation have been well characterized (Alquézar et al., 2008; Kato et al., 2004; Kaewsuksaeng et al., 2015; Rodrigo et al., 2004; Yin et al., 2016). In plants, the biosynthesis of carotenoids is catalyzed by a series of desaturation, cyclization, hydroxylation, and epoxidation enzymes, including phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), lycopene β-cyclase (LCYb), lycopene ε-cyclase (LCYe), β-ring hydroxylase (HYb), and zeaxanthin epoxidase (ZEP). Previous studies suggested that CitPSY, CitLCYb2, and CitHYb were the key genes regulating carotenoid accumulation in the flavedo and juice sacs of citrus fruit (Alquézar et al., 2009; Fujii et al., 2021; Kato et al., 2004; Ma et al., 2016, 2023; Peng et al., 2013; Zhang et al., 2012). During the ripening process, significant increases in the expression of CitPSY, CitLCYb2, and CitHYb led to massive accumulation of β,β-xanthophylls, which were the major carotenoids responsible for the yellow color of flavedo and juice sacs. As shown in Figure 1, carotenoid and chlorophyll share a common biosynthetic pathway, using geranylgeranyl diphosphate (GGPP) as a precursor. The phytol chain of chlorophyll is synthesized from the reduction of GGPP catalyzed by geranylgeranyl reductase (GGDR). In citrus fruit, metabolic flux changing from chlorophyll biosynthesis to carotenoid biosynthesis occurred during the ripening process along with rapid decreases in the expression of chlorophyll biosynthetic genes (CitGGDR, CitCHLH, CitCHLM, CitCHL27, CitPORA, CitCS, and CitCAO; Alós et al., 2006; Ma et al., 2021a, b). In addition, the degradation of chlorophylls to non-fluorescent chlorophyll catabolites catalyzed by CitCLH, CitPPH, CitPAO, and CitRCCR also caused the loss of chlorophylls during the ripening process in citrus fruit (Yin et al., 2016).

Fig. 1

Metabolic pathway involved in the metabolism of carotenoid and chlorophyll via the MEP pathway in citrus. MEP pathway, methylerthritol-4-phosphate pathway; GGPP, geranylgeranyl diphosphate. The enzymes investigated in this study were: CHLH, magnesium chelatase; CHLM, magnesium-protoporphyrin IX methyltransferase; CHL27, Mg-Proto IX monomethyl ester cyclase; PORA, protochlorophyllide oxidoreductase A; CS, chlorophyll synthase; CAO, chlide a oxygenase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase.

Auxin is one of the most important plant hormones regulating plant growth and development. In citrus, auxin has been reported to be effective to induce fruit set, stimulate plant growth, and maintain fruit quality by retarding calyx abscission during storage (Agustí et al., 2002; Bermejo et al., 2018; Carvalho et al., 2008). In previous studies, we found that auxin treatment enhanced the carotenoid accumulation and improved the poor coloration in gibberellin (GA) and prohydrojasmon (PDJ)-treated puffy fruit (Ma et al., 2021c). Moreover, the expression of ethylene biosynthetic genes and ethylene production were induced by auxin treatment, which indicated that the regulation of carotenogenesis by auxin in citrus fruit may be exerted via the stimulation of ethylene biosynthesis. ‘Miyagawa-wase’ is an early-season citrus variety widely consumed in Japan. The fruit of ‘Miyagawa-wase’ are harvested at the beginning of autumn when the pulp reaches maturity while the peel is still a greenish color. To improve the coloration of the ‘Miyagawa-wase’ fruit, the effects of postharvest treatment of 1-naphthaleneacetic acid (NAA) on chlorophyll and carotenoid metabolism were investigated in this study. In addition, to further verify the interaction between auxin and ethylene in the regulation of citrus fruit degreening, we investigated the effects of a combination treatment of NAA and 1-methylcyclopropene (1-MCP), which is an ethylene antagonist, on the degreening of citrus fruit in this study. The results will contribute to elucidating the roles of auxin in plants, and provide new insights into the improvement of coloration in early-season citrus fruit.

Materials and Methods

Plant materials and treatments

Satsuma mandarins ‘Miyagawa-wase’ (Citrus unshiu Marc.) cultivated at the Fujieda Farm of Shizuoka University (Shizuoka, Japan) were used as plant materials. Fruit of regular shape and uniform size and color were selected, and the physically damaged ones were removed. The fruit were harvested from the farm on November 1^st 2019, and dipped in a solution of 500 μM NAA for 1 h. After air-drying, half of the fruit were placed inside a 38-L airtight chamber and treated with 1 μL·L⁻¹ 1-MCP for 24 h at ambient temperature. Fruit dipped in deionized water were used as the control. After each treatment, the fruit were stored in plastic baskets covered with plastic wrap at 15°C for six days (80–90% humidity). The flavedo in each treatment was sampled on the 3^rd day and 6^th day, immediately frozen in liquid nitrogen, and stored at −80°C until use. In this study, 10 fruit were used in each treatment. Three replicates were conducted in the analysis of chlorophyll and carotenoid contents and gene expression for each treatment.

Color analysis

The fruit surface color was determined with a colorimeter (NR-12A, Nippon Denshoku, Japan) at three positions on the equatorial plane of each fruit. The CIE 1976 (L*, a*, and b*) color scale was used in this study. The hue angle (H°) was calculated as tan⁻¹ (b*/a*) and the citrus color index (CCI) was calculated as 1000 × a*/(L* × b*) according to the methods described by Ma et al. (2021b).

Chlorophyll measurement

N,N-dimethylformamide was used to assay the Chl a and b content following the methods of Moran (1982). Flavedo powder (0.5 g) was incubated overnight in 5 mL of N,N-dimethylformamide at room temperature. After being centrifuged at 3,000 rpm for 10 min, the absorbances of each sample at 664 and 647 nm were determined by spectrophotometer. The contents of chlorophyll a, chlorophyll b, and total chlorophyll were calculated according to Moran’s method and expressed as μg·g⁻¹ fresh weight.

Carotenoids measurement

The identification, extraction and quantification of carotenoids in the flavedo were carried out according to Kato’s methods (Kato et al., 2004). Pigments were extracted from the samples using an extraction solution (hexane: acetone: ethanol, 2:1:1, v/v) containing 10% (w/v) magnesium carbonate basic. After the organic solvents were completely evaporated at a maximum of 35°C under vacuum conditions, the dry samples were re-suspended in diethyl ether containing 0.1% (w/v) butylated hydroxytoluene (BHT) and saponified overnight with 20% (w/v) methanolic KOH. Then, the water-soluble extracts were removed by adding NaCl-saturated water. Anhydrous Na₂SO₄ was added to remove residual water from the sample. The carotenoids in the diethylether phase were collected and evaporated to dryness. Subsequently, the residue was redissolved in 5 mL of TBME: methanol (1:1, v/v) solution containing 0.5% (w/v) BHT. Finally, 20 μL of sample was auto-injected into a reverse-phase HPLC system (Jasco, Tokyo, Japan) fitted with a YMC Carotenoid S-5 column (Waters, Milford, MA, USA). The carotenoids contents were estimated by standard curves and expressed as μg·g⁻¹ fresh weight. The total carotenoid content was calculated by summing all identified carotenoids. Carotenoid quantification was performed using three replicates.

Analysis of gene expression by real-time quantitative RT-PCR

Total RNA was extracted from the flavedo according to Ikoma’s method (Ikoma et al., 1996). After extraction, the total RNA was purified by using an RNeasy Mini Kit (Qiagen, Hilden, Germany) incorporating on-column DNase digestion. In this study, the reactions of reverse transcription (RT) were performed at 37°C with 1.5 μg of purified RNA, a random hexamer, and TaqMan reverse transcription Reagents (Applied Biosystems, Foster City, CA, USA).

TaqMan MGB probes and sets of primers for chlorophyll metabolism genes (CitGGDR, CitCHLH, CitCHLM, CitCHL27, CitPORA, CitCS, CitCAO, CitCLH, CitPPH, CitPAO, and CitRCCR), carotenoid biosynthesis genes (CitPSY, CitPDS, CitZDS, CitLCYb1, CitLCYb2, CitLCYe, CitHYb, and CitZEP) were previously described by Kaewmanee et al. (2022). TaqMan real-time PCR was performed by using TaqMan Universal PCR Master Mix (Applied Biosystems) and a StepOnePlus^TM Real-Time PCR System (Applied Biosystems). The reaction mixture contained template cDNA, primers (900 nM), and a TaqMan MGB Probe (250 nM). The thermal cycling conditions were 95°C for 10 min, followed by 40–50 cycles of 95°C for 15 s and 60°C for 60 s. In this study, TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems) was used as endogenous control. Real-time quantitative RT-PCR was performed using three replicates for each sample.

Statistical analysis

All data are shown as the mean ± SE. The data were analyzed and Tukey’s HSD test (P < 0.05) was used to compare significant differences among different treatments.

Results

Effect of NAA on citrus fruit degreening after harvest

In this study, the fruit of ‘Miyagawa-wase’ were harvested on November 1^st when the pulp reached maturity and was ready to eat. Because of the relatively high temperature in the harvest season, the peel of ‘Miyagawa-wase’ did not develop full coloration and was still a greenish color (Fig. 2). To improve the peel coloration, the effect of NAA on degreening was investigated in ‘Miyagawa-wase’ after harvest. As shown in Figure 2, the degreening process in the peel was significantly accelerated by the NAA treatment during the storage period, and the peel of the NAA-treated fruit turned yellow on the 6^th day after harvest. The changes in the peel color were further measured by hue angle and CCI (Fig. 3). In the control, the hue angle fell from 96° to 86°, and the CCI values increased from negative values to positive values during the storage period, which indicated that fruit lost chlorophylls and exhibited a characteristic yellow color. In the NAA treatment, the changes in the hue angle and CCI value were significantly accelerated during the storage period. Compared with the control, the lower hue angle and higher CCI values in the NAA-treated fruit were consistent with the deeper yellow color in the peel on the 6^th day after harvest (Fig. 3B). In addition, we also investigated the effects of the combination treatment of NAA and 1-MCP on the degreening of citrus fruit in this study. As shown in Figure 2A, the promoting effect on degreening in the NAA treatment was inhibited by 1-MCP. In the combination treatment of NAA and 1-MCP, no significant changes in the peel coloration were observed as compared with the control. The hue angle and CCI value in the NAA and 1-MCP-treated fruit were similar to the control on the 6^th day after harvest (Fig. 3).

Fig. 2

Changes in the appearance of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP.

Fig. 3

Changes in the hue angle (A) and citrus color index (B) in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. The decrease in the hue angle (H°) indicates the peel color changing from green to yellow. A negative citrus color index (CCI) value means green peel, and a positive CCI value means yellow peel. The results shown are the means and SE (n = 6). The letters (a and b) indicate significant differences at the 5% level by Tukey’s test.

Effect of NAA on the chlorophyll content and the expression of chlorophyll metabolic genes in the flavedo of citrus fruit after harvest

Chlorophyll contents decreased rapidly in the control, NAA treatment, and NAA plus 1-MCP treatment on the 3^rd day after harvest. On the 6^th day, the chlorophyll contents continued decreasing only in the NAA treatment. Compared with the control, the NAA treatment did not affect the chlorophyll contents on the 3^rd day, while it caused significant decreases in chlorophylls in the flavedo on the 6^th day after harvest (Fig. 4). The total chlorophyll content in the NAA treatment was much lower than that of the control on the 6^th day after harvest. However, the effects of NAA treatment on chlorophyll degradation were eliminated in the fruit treated with NAA plus 1-MCP. In the combination treatment of NAA and 1-MCP, the total chlorophyll content was higher than that of the control on the 6^th day after harvest (Fig. 4).

Fig. 4

Changes in the chlorophyll content in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. Columns and bars represent the means and SE (n = 3), respectively. The letters (a, b, and c) indicate significant differences at the 5% level by Tukey’s test.

Changes in the expression of chlorophyll biosynthetic genes (CitGGDR, CitCHLH, CitCHLM, CitCHL27, CitPORA, CitCS, and CitCAO) and breakdown genes (CitCLH, CitPPH, CitPAO, and CitRCCR) were investigated in the flavedo after harvest. As shown in Figure 5, the expression of CitGGDR, CitCHLH, CitCHLM, CitCHL27, CitPORA, and CitCS decreased rapidly in the flavedo of the control during the storage period, which was consistent with the decreases in the contents of chlorophylls after harvest. On the 6^th day, the expression of CitGGDR, CitCHLH, CitCHL27, CitPORA, and CitCAO was down-regulated by the NAA treatment. In contrast, the expression levels of CitCHLH, CitCHLM, CitCHL27, CitPORA, and CitCS in the treatment of NAA plus 1-MCP were higher than those of the control and NAA treatment alone on the 6^th day after harvest. In the control, the expression of chlorophyll breakdown genes clearly decreased during the storage period, except for CitPPH. The expression of CitCLH and CitPPH were up-regulated by the NAA treatment on the 6^th day after harvest (Fig. 6). Compared with the NAA treatment alone, the expression level of CitPPH was lower in the combination treatment of NAA and 1-MCP on the 6^th day after harvest.

Fig. 5

Changes in chlorophyll biosynthetic genes’ expression in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. Columns and bars represent the means and SE (n = 3), respectively. The letters (a, b, and c) indicate significant differences at the 5% level by Tukey’s test.

Fig. 6

Changes in chlorophyll degradation genes’ expression in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. Columns and bars represent the means and SE (n = 3), respectively. The letters (a, b, and c) indicate significant differences at the 5% level by Tukey’s test.

Effect of NAA on the carotenoid content and the expression of carotenoid biosynthetic genes in the flavedo of citrus fruit after harvest

Changes in the contents of β-carotene, β-cryptoxanthin, all-trans-violaxanthin, 9-cis-violaxanthin, and lutein were investigated in the flavedo after harvest. During the storage period, β-cryptoxanthin and 9-cis-violaxanthin, which are the major carotenoids responsible for the yellow color of the mature fruit, accumulated gradually in the flavedo. In the NAA treatment, the contents of β-cryptoxanthin and 9-cis-violaxanthin were significantly increased on the 6^th day after harvest, which were 1.6 times of the control (Fig. 7). In the combination treatment of NAA and 1-MCP, the contents of β-cryptoxanthin and 9-cis-violaxanthin were also slightly increased, but their contents were lower compared with NAA treatment alone. In addition, the lutein content was decreased by the NAA treatment on the 6^th day after harvest, but was not significantly affected by the NAA plus 1-MCP treatment (Fig. 7).

Fig. 7

Changes in carotenoid content in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. β-Car, β-carotene; β-Cry, β-cryptoxanthin; Lut, lutein; T-vio, all-trans-violaxanthin; C-vio, 9-cis-violaxanthin; Total, total carotenoid. The total carotenoid was calculated by summing β-carotene, β-cryptoxanthin, lutein, all-trans-violaxanthin, and 9-cis-violaxanthin. Columns and bars represent the means and SE (n = 3), respectively. The letters (a, b, and c) indicate significant differences at the 5% level by Tukey’s test.

The expression of carotenoid metabolic genes (CitPSY, CitPDS, CitZDS, CitLCYb1, CitLCYb2, CitLCYe, CitHYb, and CitZEP) was analyzed. In the control, the expression of all the genes investigated increased gradually during the storage period, except for CitLCYe, which decreased rapidly after harvest. In the NAA treatment, the expression of carotenoid biosynthetic genes CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb was up-regulated on the 6^th day after harvest (Fig. 8). Compared with NAA treatment alone, the expression of CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb was down-regulated in the combination treatment of NAA and 1-MCP on the 6^th day after harvest.

Fig. 8

Changes in carotenoid biosynthetic genes’ expression in the flavedo of citrus fruit after postharvest treatment with NAA or NAA + 1-MCP. Columns and bars represent the means and SE (n = 3), respectively. The letters (a, b, and c) indicate significant differences at the 5% level by Tukey’s test.

Discussion

Citrus fruit degreening is closely associated with falls in seasonal temperatures, and low over-night temperatures (below 13°C) are required to stimulate the color break of citrus fruit on trees (Mitalo et al., 2020). ‘Miyagawa-wase’ is an early-season citrus variety that is harvested at the beginning of autumn in Japan. When the pulp reaches maturity, the peel is still green because of the relatively higher seasonal temperature when the fruit of ‘Miyagawa-wase’ is harvested. In citrus, a bright and attractive yellow peel is an important index of fruit quality and consumer acceptance, while a greenish appearance is often associated with unripe fruit. Therefore, the practice of degreening after harvest is especially important for ‘Miyagawa-wase’ to improve the peel coloration and render the fruit attractive to consumers. Auxin is an important plant hormone regulating plant development and growth. In our previous study, we found that auxin treatment was an effective method to improve the poor coloration of GA and PDJ-treated puffy fruit (Ma et al., 2021c). In the present study, to accelerate the degreening of ‘Miyagawa-wase’ peel, we treated the fruit with NAA after harvest. The results showed that the coloration in the peel was significantly improved by the NAA treatment during the storage period. On the 6^th day after harvest, the peel color of the NAA-treated fruit turned yellow with higher CCI values than the control (Fig. 3). This result indicated that postharvest auxin treatment is a promising approach to improve the peel coloration in early-season citrus varieties during storage.

The degreening of citrus fruit is accompanied by decreases in chlorophylls and increases in carotenoids. In citrus, the key genes involved in chlorophyll and carotenoid metabolism have been isolated and well characterized (Alquézar et al., 2008; Cazzonelli and Pogson, 2010; Kaewsuksaeng et al., 2015; Kato et al., 2004; Rodrigo et al., 2004; Yin et al., 2016). During degreening, the transcriptional regulation of chlorophyll and carotenoid metabolic genes is a major mechanism that modulates the chlorophyll and carotenoid accumulation in citrus fruit. In this study, the results showed that the reduction of the chlorophyll contents during degreening was accompanied by decreases in the expression of chlorophyll biosynthetic genes (CitGGDR, CitCHLH, CitCHLM, CitCHL27, CitPORA, and CitCS) and increases in the chlorophyll degradation gene CitPPH in the control. In the NAA treatment, the expression of CitGGDR, CitCHLH, CitCHL27, CitPORA, and CitCAO) was down-regulated, while the expression of CitPPH was up-regulated, on the 6^th day after harvest (Figs. 5 and 6). The lower expression levels of chlorophyll biosynthetic genes and higher expression level of chlorophyll degradation genes led to accelerated loss of chlorophyll in the NAA treatment.

In citrus fruit, it was reported that carotenoid biosynthesis changed from the ε,β-branch to β,β-branch in the flavedo of citrus fruit during the ripening process. With the transition from the green stage to yellow stage, the lutein content decreased, while the contents of β-cryptoxanthin and 9-cis-violaxanthin increased in the flavedo of citrus fruit (Kato et al., 2004; Zhang et al., 2012). Here, the results showed that the NAA treatment accelerated the shift from the ε,β-branch to β,β-branch in the carotenoid biosynthesis in ‘Miyagawa-wase’. Compared with the control, the lutein content was lower, while the contents of β-cryptoxanthin and 9-cis-violaxanthin were higher, in the NAA treatment on the 6^th day after harvest (Fig. 7). Gene expression results showed that the expression of CitLCYe was down-regulated by the NAA treatment, which was consistent with the lower lutein content in the NAA-treated fruit on the 6^th day after harvest. In contrast, the expression of CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb was significantly up-regulated by the NAA treatment on the 6^th day after harvest (Fig. 8). The high expression of genes related to β,β-carotenoid biosynthesis, especially CitLCYb2 and CitHYb, led to increased contents of β-cryptoxanthin and 9-cis-violaxanthin in the NAA-treated fruit. These results suggested that postharvest NAA treatment was effective to induce chlorophyll degradation and carotenoid accumulation in ‘Miyagawa-wase’ during storage.

Auxin physiologically interacts with ethylene in plants, and exogenous auxin treatment stimulates ethylene biosynthesis by inducing the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. This interaction between auxin and ethylene is important for the regulation of plant growth and development (Robles et al., 2013; Stepanova et al., 2007; Yamauchi et al., 2020). In Arabidopsis, auxin is a positive regulator for the ethylene-mediated response, and it was required for the ethylene-mediated growth response in the roots (Rahman et al., 2001). In our previous study, it was found that postharvest auxin treatment induced the expression of ethylene biosynthetic genes and ethylene production in GA and PDJ-treated puffy citrus fruit, which indicated that the regulation of carotenoid accumulation by auxin may be caused by ethylene in citrus fruit (Ma et al., 2021c). To further confirm this, we treated the fruit with a combination of NAA and the ethylene antagonist 1-MCP in this study. The result showed that the positive effects of NAA treatment on chlorophyll degradation were eliminated in the combination treatment with NAA and 1-MCP on the 6^th day after harvest. In the combination treatment of NAA and 1-MCP, the total chlorophyll content was much higher than that in the NAA treatment, which was consistent with the up-regulation of chlorophyll biosynthetic genes (CitCHLH, CitCHLM, CitCHL27, CitPORA, and CitCS) and downregulation of the chlorophyll degradation gene CitPPH. Compared with the NAA treatment alone, the expression of carotenoid biosynthetic genes, CitPSY, CitPDS, CitZDS, CitLCYb2, CitLCYe and CitHYb was down-regulated by the combination treatment of NAA and 1-MCP, which led to lower contents of β-cryptoxanthin and 9-cis-violaxanthin in the NAA and 1-MCP-treated fruit on the 6^th day after harvest. These results suggest that there is an interaction between auxin and ethylene in the regulation of chlorophyll and carotenoid accumulation in citrus fruit, and the acceleration of degreening by exogenous auxin is caused by ethylene in citrus fruit.

In conclusion, the results presented in this study showed that NAA treatment accelerated chlorophyll reduction and carotenoid accumulation in the peel after harvest, which was caused by the induction of endogenous ethylene generation in citrus fruit. These results indicate that postharvest NAA treatment is an effective strategy to improve the coloration and enhance the commercial value of early-season citrus varieties.

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