Regulation of Chlorophyll and Carotenoid Metabolism in Citrus Fruit During Maturation and Regreening

Abstract

Chlorophyll and carotenoid are the main pigments that are responsible for coloration of citrus fruit. The changes in their contents are related to the development stage in citrus fruit. During fruit maturation, carotenoids are massively accumulated in the flavedo and juice sacs, while the chlorophyll contents decreased rapidly. In contrast, the increase of chlorophyll content and decrease of carotenoid contents are observed in the fruit during regreening process. In addition, plant hormones and environmental conditions play important roles in the regulation of color development and the changes of chlorophyll and carotenoid contents in citrus fruit. In this review, we summarized the changes in chlorophyll and carotenoid contents in the flavedo and juice sacs of citrus fruit during fruit maturation and regreening process. Current understanding on the molecular mechanisms that regulated the chlorophyll and carotenoid metabolisms in citrus fruit are discussed. Moreover, the effects of plant hormones and environmental conditions on chlorophyll and carotenoid accumulation are also presented in this review.

1. Introduction

Citrus is one of the most popular and main fruit crops in the world. Citrus fruit is a rich source of vitamins, minerals, and dietary fiber that are essential for overall human health. In most citrus varieties, fruit coloration is attributed to the massive accumulation of chlorophylls and carotenoids. Chlorophylls are predominantly accumulated in the immature fruit and responsible for the green color in the peel of citrus fruit. Carotenoids are responsible for the characteristic coloration of the mature fruit. Because of the different carotenoid content and composition, the fruit color varies greatly among different citrus varieties, which ranges from bright yellow color in lemon, grapefruit, and pummelo, to characteristic orange color in mandarins and sweet oranges, or the red color in grapefruit and pummelos. In some varieties of citrus fruit, when the fruit is left on the tree till the spring or summer season, the color of the fruit will reverse from orange to green during fruit maturation, and this process is called “regreening” of the citrus fruits. The coloration is an important index of the commercial and nutritional values of citrus fruit, and it influences the perceptions of the consumers. To improve the fruit coloration, extensive researches have been done on the chlorophyll and carotenoid metabolisms, and significant process has been made in the elucidation of chlorophyll and carotenoid accumulation in citrus fruit in the past decades. Plant hormones and environmental conditions are important regulators of color development in citrus fruit. Plant hormones, such as ethylene, auxin, abscisic acid (ABA), and jasmonate (JA) and environmental conditions (low temperature and red light) accelerated the fruit degreening by reducing chlorophyll contents and stimulating the carotenoid accumulation in flavedo during fruit maturation. In addition to the above factors, gibberellin (GA) and high temperature have negative effects on fruit coloration by inhibiting the chlorophyll breakdown and carotenoid biosynthesis in fruit flavedo. Moreover, the reduction of carotenoid accumulation and re-accumulation of chlorophyll are induced by GA, high temperature, and blue light in the flavedo during the regreening process. In this review, we summarized the recent researches on chlorophyll and carotenoid accumulation and regulation in citrus fruit during the maturation and regreening processes. It will contribute to a better understanding on chlorophyll and carotenoid metabolisms, and provide new insights into improving the coloration of citrus fruit.

2. Pigment in citrus fruits

2.1 Chlorophylls

2.1.1 Chlorophyll metabolism pathway in citrus fruit

As shown in Fig. 1, the biosynthesis of chlorophyll and carotenoid shares a common precursor geranylgeranyl diphosphate (GGPP). Chlorophylls are synthesized in plastids (chloroplasts) and are mediated by many nuclear-encoded enzymes. There are condensed from chlorophyllide (a) and phytyl-PP. Chlorophyllide (a) is synthesized from glutamate in plastids, and phytyl-PP is produced from GGPP catalyzed by geranylgeranyl-PP reductase (GGDR) (Fig. 1). The condensation of phytyl-PP and 3-vinyl-chlorophyllide a to form chlorophyll a (Chl a) is catalyzed by chlorophyll synthase (CS). Subsequently, Chl a can be converted into chlorophyll b (Chl b) by chlorophyll a oxygenase (CAO) [1, 2, 3, 4]. The chlorophyll degradation starts with the conversion of Chl b to Chl a by Chl b reductase (NYC), and then the Chl a is converted to chlorophyllide a by chlorophyllase (CLH) to remove of the side chain attached to the tetrapyrrole macrocycle. Afterwards, the magnesium in the center of chlorophyllide is removed by action of magnesium dechelatase (STAY-GREEN, SGR), thereby producing pheophytin a, which is then catabolized to pheophorbide a and free phytol by the action of pheophorbide hydrolase (PPH). Finally, pheophorbide a is converted to the “red chlorophyll catabolite” (RCC) by the action of pheophorbide a oxygenase (PAO), followed by conversion of RCC into “fluorescent chlorophyll catabolites” (FCCs) by red chlorophyll catabolite reductase (RCCR) [1, 2, 5, 6, 7].

2.1.2 Changes in chlorophyll accumulation during citrus fruit maturation

Chlorophylls are the main pigments in the immature flavedo, representing green color. When the color break occurs in citrus fruit, the contents of chlorophylls start to decrease progressively and virtually disappear in the peel of fully colored fruit [2]. There are two types of chlorophyll, Chl a and Chl b, accumulated in citrus fruit, and Chl a is the major component in the citrus flavedo [8, 9, 10]. During the fruit maturation, the decrease in chlorophyll accumulation was accompanied by the decrease of the gene expression of the GGDR and increase of the expression of chlorophyll degradation genes (NYC, PPH, CLH1, SGR1, and ACD2) in the flavedo of citrus fruit [11, 12, 13] (Fig. 2).

2.1.3 Changes in chlorophyll accumulation during citrus fruit regreening

Regreening is a phenomenon that the flavedo of fruit regains green color when left the fruit on the tree till the summer season [14]. In citrus fruit, the regreening process was investigated in Valencia orange, which is a late-season variety. During the regreening, chlorophylls were re-accumulated in the peel, and the peel color changed from orange to green. Moreover, the regreening fruit contained higher contents of chlorophylls in the stem end area than in the apical and equatorial area of the peel [15, 16]. It was reported that the reversion of chromoplast to chloroplast occurred during the regreening [17, 18]. In addition, the contents of Chl a, Chl b, and total chlorophyll were simultaneously increased in the fruit flavedo, especially in the top part [16] (Fig. 2). The regreening of citrus fruit was regulated by promoting the chlorophyll biosynthesis and repressing the chlorophyll degradation at the transcriptional level. The increase in the expression of chlorophyll biosynthetic genes (GGDR, CHL27, PORA, and CAO) and the decrease in the expression of chlorophyll degradation genes (CLH1, SGR, PPH, PAO, and RCCR) contributed to increasing chlorophylls contents in the regreened fruit.

Figure 1: Metabolic pathways involved in biosyntheses of carotenoid and chlorophyll via the MEP pathway in plants. MEP pathway, methylerthritol-4-phosphate pathway; GGPP, geranylgeranyl diphosphate. The enzymes investigated in this study are: PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotene isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYb, β-ring hydroxylase; HYe, ε-ring hydroxylase; ZEP, zeaxanthin epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase; VDE, violaxanthin de-epoxidase; CCD; carotenoid cleavage dioxygenase; GGDR, geranylgeranyl reductase; 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 oxygeanase; NYC, non-yellow coloring; CLH, chlorophyllase; SGR, Stay-Green; PPH, pheophytin pheophorbide hydrolase; PAO, pheophorbide a oxygenase; RCCR, Red chlorophyll catabolite reductase.

2.2 Carotenoids

2.2.1 Carotenoid metabolism pathway in citrus fruit

Carotenoids are synthesized in plastids (chloroplasts and chromoplasts), and are mediated by many nuclear-encoded enzymes (Fig. 1). The first committed step of carotenoid biosynthesis is condensed of two molecules of GGPP to ultimately form colorless phytoene (C₄₀) by the action of phytoene synthase (PSY). Then the desaturation reaction of phytoene is catalyzed by phytoene dehydrogenase (PDS) and ξ-carotene desaturase (ZDS), which introduce four double bounds into phytoene to yield lycopene via phytofluene, 𝜁-carotenoid, and neurosporene. Lycopene molecules are standing at a branching point leading either to α-carotene or to β-carotene, depending on two different cyclases. Lycopene β-cyclase (LCYb) and lycopene ε-cyclase (LCYe) are responsible for these reactions. Interestingly, it had been reported that the LCYb activity was restricted to chromoplastic tissues and only active in the production of β,β-carotenoids [19, 20, 21]. α-Carotene is converted to lutein catalyzed by ε-ring hydroxylase (HYe) and β-ring hydroxylase (HYb). β-Carotene is converted to β-cryptoxanthin and zeaxanthin via a two-step hydroxylation by HYb. In addition, the conversion from zeaxanthin to violaxanthin is catalyzed by zeaxanthin epoxidase (ZEP). In certain conditions, violaxanthin molecules can be reversible reaction into zeaxanthin by violaxanthin de-epoxidase (VDE), called xanthophylls cycle. At the last step, violaxanthin is converted to xanthoxin (C₁₅) catalyzed by 9-cis-epoxycarotenoid dioxygenases (NCEDs), and NCED2 and NCED3 had been reported in flavedo and juice sacs of citrus fruit [22]. Xanthoxin can be used to form the phytohormone ABA [2, 6, 22, 23, 24]. Moreover, carotenoids are precursor of apocarotenoids, and some of them are volatile aroma-related compounds, which is catalyzed by carotenoid cleavage dioxygenases (CCDs) [25, 26, 27]. In citrus, CCD1 is expressed in both flavedos and juice sacs, while CCD4 is specifically expressed in the flavedos of a few citrus varieties and associated with the β-citraurin biosynthesis (a red pigment) [22, 28].

2.2.2 Changes in carotenoid accumulation during citrus fruit maturation

In citrus fruit, the carotenoid composition in the flavedo is different according to the fruit mature stage. In the immature stage, high levels of β,ε-carotenoids (α-carotene and lutein) are accumulated in the flavedo, which is in parallel with the high expression of LCYe and low expression of PSY, ZDS, and HYb [23, 29]. Along with the fruit maturation, the expression of PSY, PDS, ZDS, LCYb, HYb, and ZEP is simultaneously up-regulated, which led to the massive accumulation of β,β-xanthophylls (β-cryptoxanthin and violaxanthin) in the flavedo in the mature stage [29]. In addition, the carotenoid composition in the juice sacs varies greatly among different citrus varieties. Violaxanthin is massively accumulated in the juice sacs of sweet oranges (Citrus sinensis Osbeck), such as Valencia orange, Navelina, Hamlin, and Salustiana [29]. In mandarins (C. reticulata, C. unshiu, and C. clementiana), β-cryptoxanthin was predominantly accumulated as the major carotenoid in the juice sacs. In a few red-flesh citrus varieties, such as pumelo (C. grandis Osbeck), Cara Cara orange and Hong Anliu (C. sinensis Osbeck), and grapefruit (C. paradisi Macf.), lycopene was massively accumulated in the juice sacs, and the accumulation of lycopene led the juice sacs to exhibit reddish color [20, 30, 31, 32, 33, 34]. In citrus fruit, the transcriptional regulation is a major mechanism that controls the carotenoid accumulation in the juice sacs. The expression of downstream genes (LCYb1, LCYb2, LCYe, HYb, and HYe) was higher in the juice sacs of oranges and mandarins, which was consistent with the accumulation of violaxanthin and β-cryptoxanthin. In contrast, the expression of the LCYb1, LCYb2, LCYe, HYb, and HYe was lower in the juice sacs of red-flesh citrus varieties, and the low expression of the downstream genes led to the massive accumulation of lycopene in the juice sacs.

2.2.3 Changes in carotenoid accumulation during citrus fruit regreening

In addition to the chlorophyll, the carotenoid accumulation was changed during the fruit regreening process, which was accompanied with the reversion of chromoplasts to chloroplasts and the formation of new chloroplasts [35]. The reversion of chromoplasts to chloroplasts occurred with the disappearance or reduction in size and number of plastoglobules and the formation of new thylakoids or reformation of thylakoids, leading to normal chloroplast structure and photosynthetic activity [17, 36, 37, 38]. In citrus fruit, along with the decrease in the number of chromoplasts, the accumulation of carotenoid was reduced. The contents of β,β-carotenoids (β-cryptoxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin) and the total carotenoids in the flavedo of citrus fruit decreased during regreening process. In the meanwhile, the expression of carotenoid biosynthetic genes (PSY, PDS, ZDS, LCYb2, and HYb) was significantly down-regulated in the flavedo, which was consistent with the reduction of carotenoid during the regreening process [16] (Fig. 2).

Figure 2: Changes in chlorophyll and carotenoid contents and expression of genes involved in pigment metabolism of citrus fruit. Chl a, chlorophyll a; Chl b chlorophyll b; α-Car, α-carotene; Lut, lutein; β-Car, β-carotene; β-Cry, β-cryptoxanthin; Zea, zeaxanthin; Vio, violaxanthin.

3. Factors affecting chlorophyll and carotenoid accumulation

3.1 Plat hormones

3.1.1 Ethylene

Ethylene is a common plant hormone with numerous influences in fruit crops. According to the ripening behavior, fruit is classified into two types, climacteric and non-climacteric fruit. In climacteric fruit, ethylene is the main hormone controlling the fruit maturation, and the burst of autocatalytic ethylene co‐ordinates and accelerates the maturation process. On the contrary, the non-climacteric fruit, such as citrus fruit, grape, and strawberry, produces a low level of ethylene during the maturation process. In citrus fruit, previous studies indicated that the carotenoid accumulation and chlorophyll degradation in the flavedo are induced by ethylene [39, 40, 41, 42]. In lemon and sweet orange, ethylene treatment decreased the chlorophylls contents and accelerated the degreening in the peel. In the ethylene treatment, the accumulation of phytoene, phytofluene, 9-cis-violaxanthin, and β-citraurin was induced and the expression of carotenoid biosynthetic genes was up-regulated [2, 40]. In Yamashitabeni-wase, which is a variety of Satsuma mandarin, the accumulation of the red pigment β-citraurin was enhanced by the ethylene treatment along with the increase of the expression of CCD4 in the flavedo [28]. In the studies of Mitalo et al. [12, 13], it was found that the expression of the genes associated with chlorophyll degradation (CLH1 and PPH), carotenoid biosynthesis (PSY1, LCYb2a, and NCED5), and transcription factors (ERF114 and bHLH25) was dramatically induced in response to the ethylene treatment. Moreover, the genes associated with chlorophyll degradation (such as SGR1, NOL, and ACD2) were differentially regulated by propylene (a well-known ethylene analog) in flavedo of Satsuma mandarin. In addition, the ethylene and ethylene analog promoted the fruit coloration by activating the ethylene-specific TFs, including ERF5, ERF6, ERF7, ERF13, ERF114, and ERF061 [11, 12, 13, 43, 44, 45]. Among them, ERF5, ERF6, ERF7, ERF114, and ERF13 negatively regulated chlorophyll accumulation in citrus fruit. In previous studies, it was revealed that the activation of the ERF6 and ERF13 promoters and the subsequent automatic and mutual regulation between ERF6 and ERF13 led to the increase of the expression of chlorophyll degradation-related gene PPH [11, 44]. Mitalo et al. [12] reported that ERF114 activated the expression of chlorophyll degradation genes (CLH1 and PPH), and caused a decrease of chlorophyll content in the peel. In the study of Zhu et al. [45], it was found that ERF061 enhanced carotenoid accumulation by directly regulating a series of key carotenoid metabolic genes (PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4).

3.1.2 Auxin

Auxin, an important plant hormone, interacts with ethylene to regulate the biosynthesis of secondary metabolites. The recent research reported that the two auxins, indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA, synthetic auxin) were effective to enhance carotenoid accumulation in both flavedos and juice sacs of Satsuma mandarin ‘Aoshima unshiu’. Kato [46] reported that the postharvest treatment of NAA improved carotenoid accumulation in concentration-dependent manner. Along with the increases of NAA concentration, there was a gradual increase of carotenoid contents in the flavedo of citrus fruit. In the treatment of NAA, the accumulation of β-carotene, β-cryptoxanthin, lutein, all-trans-violaxanthin, and 9-cis-violaxanthin was remarkably stimulated along with the up-regulation of the expression of carotenoid biosynthetic genes (PSY, ZDS, LCYb1, LCYb2, LCYe, HYb, HYe, and ZEP) in the flavedo. Moreover, storage temperature at 15 °C and red LED light irradiation could enhance the positive effects of NAA on carotenoid accumulation in citrus fruit after harvest. In citrus, the spraying of a combination of GA and prohydrojasmon (PDJ) on tree at the color break stage could prevent the peel puffing. However, the combination treatment of GA and PDJ caused a delay in the accumulation of carotenoid and led to the mature fruit with poor color in citrus fruit [3, 4]. It was found that the preharvest treatment of NAA and postharvest treatment of IAA and NAA can improve the GA and PDJ-treated fruit coloration by reducing the chlorophylls contents and enhancing the β,β-xanthophylls contents in the flavedo [3, 4, 46]. Moreover, the modification of the chlorophyll and carotenoid metabolisms was highly regulated at the transcriptional level. In addition, the endogenous ethylene production and the expression of ethylene biosynthetic genes (ACS1, ACS2, and ACO) were induced by the IAA and NAA treatments in citrus fruit. Thus, we deduced that the regulation of chlorophyll and carotenoid accumulation in citrus fruit by auxin might be exerted through endogenous ethylene.

3.1.3 Gibberellin

GA has been applied to retard the ripening and senescence in many fruits. In citrus, Fujii et al. [47] reported that GA treatment reduced the expression of carotenoid metabolic genes and enhanced the expression of Mg-chelatase gene, which led to a delay in degreening of the fruit. In the study of Alós et al. [48], it was found that the expression of the gene encoding chlorophyll-degrading pheophorbide an oxygenase (PAO) was repressed by the GA treatment in citrus fruit during fruit degreening. In addition, the GA treatment delayed the metabolic flux changing from β,α-carotenoid biosynthesis to β,β-carotenoid biosynthesis, and led to the accumulation of lutein in the citrus flavedo [40, 48, 49]. Similar to the flavedo, the GA treatment also caused a significant decrease in the β,β-carotenoid accumulation in the juice sacs. Zhang et al. [50] reported that the expression of LCYb1 and LCYb2 was down-regulated by the GA treatment, which led to a significant decrease in the contents of β-cryptoxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin in the juice sacs of Satsuma mandarin. In Valencia orange, the expression of carotenoid biosynthesis genes (PSY, PDS, ZDS, LCYb2, HYb, and ZEP) was simultaneously down-regulated by the GA treatment, and the contents of β-carotene, lutein, all-trans-violaxanthin, and 9-cis-violaxanthin were decreased in the juice sacs. Recently, the isoprothiolane (diisopropyl-1, 3-dithiolan-2-ylidenemalonate: IPT), which is a fungicide used for the control of rice blast, was applied to use for improving peel color in Satsuma mandarin before harvest [51, 52, 53]. The IPT treatment decreased GA concentration in citrus fruit by inhibiting the expression of GA biosynthesis genes (GA20ox1 and GA3ox), and led to the decrease of chlorophyll content and increase of β-cryptoxanthin content in citrus fruit.

In addition, GA is also an important hormone regulating regreening in citrus fruit. Spraying with GA on the tree promoted regreening in Valencia orange. The contents of chlorophylls (the Chl a, Chl b, and total chlorophyll contents) were increased, whereas the carotenoids components (β-cryptoxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin) were decreased in the flavedo of Valencia oranges after the GA treatment. Correspondingly, the changes in the chlorophyll and carotenoid accumulation in the GA treatment were highly regulated at the transcriptional level. The up-regulation of chlorophyll biosynthesis genes (GGDR, CHL27, PORA, and CAO) and down-regulation of degradation genes (CLH1, SGR, PPH, PAO, and RCCR) led to the increase of chlorophyll contents, and the down-regulation of carotenoid biosynthesis genes (PSY, PDS, ZDS, LCYb2, and HYb) led to the decrease of carotenoids contents [16].

3.1.4 Abscisic acid

ABA is an end product in the carotenoid metabolic pathway, and its biosynthesis is closely related to carotenoid metabolism. In citrus fruit, ABA plays an important role in regulating fruit coloration and ripening [54, 55, 56]. Rodrigo et al. [54] investigated the role of ABA in the regulation of fruit ripening in Pinalate, which was a mutant of Navelate orange with a particle blockage in the desaturation of ζ-carotene and reduced level of ABA in both peel and pulp. The results showed that endogenous ABA played a crucial role in the regulation of fruit coloration, and the lower level of ABA led to delay the degreening in Pinalate. In addition, the ABA treatment could accelerate the fruit ripening by improving fruit color and increasing carotenoid contents in the flavedo of citrus fruit [55, 56]. In a previous study, it was reported that the preharvest spray of ABA was effective to improve the coloration in the peel of the GA and PDJ-treated fruit during fruit ripening. In the ABA treatment, the accumulation of β,β-xanthophylls was significantly induced, and the chlorophylls contents were gradually decreased in the flavedos during the ripening process. Compared with the control, the expression levels of PDS, ZDS, LCYb2, HYb, and ZEP were higher in the ABA treatment. In contrast, the expression levels of GGDR, CHLH, CHLM, CHL27, PORA, CS, and CAO in the ABA treatment were significantly lower than those of the control [3].

3.1.5 Jasmonate

Jasmonate (JA) or methyl jasmonate (MeJA) is reported to be effective to accelerate fruit ripening, enhances fruit color development, and induces the accumulation of phytonutrients in several fruits [57, 58, 59]. However, the research on the effects of JA on fruit coloration in citrus fruit is still limited. Rehman et al. [60] reported that the preharvest spray application of MeJA induced carotenoid accumulation and accelerated fruit coloration in flavedo of M7 Navel orange. In the study of Yue et al. [61], it was found that the postharvest exogenous MeJA treatment promoted the β-citraurin production and fruit coloration in Newhall orange by inducing the expression of the transcription factor MYC2, which substantially activated the expression of the key β-citraurin biosynthetic genes (CCD4b, PSY, LCYB, and HYb). Different from the flavedo, MeJA negatively regulated carotenoid accumulation of the juice sacs of citrus fruit. The MeJA treatment down-regulated the expression of HYb, and caused a significant decrease of all-trans-violaxanthin, 9-cis-violaxanthin, lutein, and total carotenoid in the juice sacs of Satsuma mandarin in vitro [62].

3.2 Environmental factors

3.2.1 Temperature

Temperature is one of the most important environmental factors that strongly affect the coloration in citrus fruit [63, 64, 65]. Most citrus fruits started to degreening when the ambient temperature decreased at the field. Low temperature at night time (below 13 °C) and moderated temperature at day time (20 °C) stimulated the fruit color break along with the decrease of chlorophylls and increases of β,β-xanthophylls in the flavedo of citrus fruit [12, 64, 66, 67, 68]. The peel degreening at low temperature was transcriptionally regulated by the activation of genes associated with chlorophyll degradation (PPH), carotenoid metabolism (PSY1, LCYb2a, CHYb1, and NCED5), photosystem protein (LHCB2), and transcription factors (ERF3 and bHLH25) [12, 13]. In addition, low/intermediate storage temperature is a key factor regulating the peel coloration of citrus fruit after harvest. In Navelina orange, Huyou, and grapefruit, the peel coloration was obviously enhanced when the fruit was stored at 12 °C and 15 °C [34, 69, 70]. In lemon, peel degreening was the most evident at 15 °C, followed by 10 °C, 20 °C, and 5 °C [12]. In contrast to low temperature, high temperature during the day has a negative effect on fruit coloration. High temperature (above 25 °C) inhibited the chlorophyll breakdown and carotenoid biosynthesis in citrus fruit [63, 71, 72]. That is why the citrus fruit grown in the tropics area is greenish peel color. Moreover, when the fruit remained on tree until spring or summer season, the peel color will turn from orange to green along with the increase of the temperature of soil and air in Valencia orange [49]. During the regreening, the expression of chlorophyll biosynthetic genes was up-regulated, while the expression of chlorophyll breakdown genes was down-regulated, which led to the peel re-accumulated chlorophylls and exhibited green peel color in late spring or summer [16].

3.2.2 Light

In citrus fruit, the external coloration is highly regulated by light. The fruit growing outside the canopy exhibited the brighter orange color as compared with the fruit growing inside the canopy [73, 74, 75]. In sweet oranges and mandarins, the direct exposure of fruit to light enhanced the carotenoid accumulation and promoted the coloration of the peel. Tao et al. [76] reported that the carotenoid accumulation was inhibited by the bagging treatment, and the carotenoid accumulation was recovered, the contents of carotenoids, especially β-cryptoxanthin, were increased when the bags were removed. In addition to the light intensity, the carotenoid accumulation was affected by the light quality. Previous studies suggested that postharvest treatment with red light was effective to enhance the carotenoid contents in fruit of kumquat, Satsuma mandarin, and Gonggan mandarin [77, 78, 79, 80]. In Satsuma mandarin, the content of β-cryptoxanthin was significantly increased and the expression of carotenoid biosynthetic genes (PSY, PDS, ZDS, LCYb1, LCYb2, HYb, and ZEP) was up-regulated by the red-light treatment [77, 78]. In the studies of Gong et al. [79, 80], it was reported that red LED light irradiation activated the expression of NAC22, which was a transcriptional factor and positively regulated the expression of LCYB1 and HYb under the red light. In addition to the ripening process, light is also a crucial factor regulating the regreening in citrus fruit. Ma et al. [81] reported that the blue LED light treatment enhanced the chlorophyll contents and induced regreening in the flavedo of Valencia orange in vitro. Under the blue light, the contents of chloroplast carotenoids, lutein, β-carotene, and all-trans-violaxanthin increased, while the contents of chromoplast carotenoid 9-cis-violaxanthin decreased during the regreening process along with the up-regulation of LCYe and down-regulation of LCYb2.

Figure 3: Regulation of exogenous hormones and environmental conditions on carotenoid content in mature fruit (A) and chlorophyll content in regreening fruit (B).

4. Conclusions

In the present study, the recent research on the chlorophyll and carotenoid accumulation and regulation was reviewed. During the fruit maturation process, chlorophyll content decreased while carotenoid increased gradually in citrus fruit. Interestingly, the re-accumulation of chlorophylls and regreening occurred in mature fruit when left on tree until summer. In citrus fruit, the accumulation of chlorophyll and carotenoid was highly regulated at the transcriptional level in both the maturation and regreening processes (Fig. 2). Moreover, the plant hormones and environmental conditions played a key role in regulating chlorophyll and carotenoid metabolisms in citrus fruit (Fig. 3). During the maturation process, plant hormones (ethylene, auxin, ABA, and JA) and environmental conditions (low temperature and red light) were effective to stimulate the carotenoid accumulation. On the other hand, the re-accumulation of chlorophyll in the flavedo was induced by GA, high temperature, and blue light during the regreening process. This review contributes to a better understanding of chlorophyll and carotenoid accumulation in citrus fruit, which will provide new strategies to improve the coloration and commercial values of citrus fruit.

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