Postharvest Biology and Technology 180 (2021) 111594

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Accumulation of tocopherols and transcriptional regulation of their

biosynthesis during cold storage of mandarin fruit
Florencia Rey, María Jesús Rodrigo, Lorenzo Zacarias *
Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avenida Agustín Escardino 7, 46980, Paterna,
Valencia, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Tocopherols are plant-derived isoprenoids with potent antioxidant activity, which have been implicated in the
Antioxidant tolerance of plants to different stresses. However, tocopherol accumulation and biosynthesis in fruit, and their
Chilling injury potential implication in postharvest chilling injury (CI), has been scarcely studied. Therefore, in this work, we
Gene expression
have investigated tocopherol accumulation and biosynthesis in the peel of mandarin fruit of three cultivars with
Mandarin
Tocopherols
contrasting susceptibility to CI during storage at 2 ◦ C (‘Fortune’>‘Nova’> ‘Nadorcott’). α- and γ-tocopherol were
the isoforms detected in the flavedo of the fruit, and a direct relationship between tocopherols content and CI-
tolerance was found, since CI-tolerant fruit accumulated the highest tocopherol content whereas CI-sensitive fruit
the lowest. Moreover, the transcriptional profiling of 14 genes related to the specific steps of tocopherol
biosynthesis, and to their precursor’s synthesis, were analyzed. Upstream genes DXS1 and DXS2 (1-deoxy-D-
xylulose-5-phosphate synthase) and GGDR (geranylgeranyl diphosphate reductase), involved in the supply of
phytyl pyrophosphate, and the VTE3 (2-methyl-6-phytyl-1,4-benzoquinol methyltransferase) isoforms appear to
be key for the differences in total tocopherol content among the cultivars at harvest. During cold storage, most
genes involved in the precursors supply were up-regulated, whereas genes of the tocopherol-core pathway were
in general repressed. Changes in VTE4 during cold storage may account for the differences in γ-tocopherol among
cultivars. Collectively, results suggest that the concentration of tocopherols at harvest may play a function in the
natural tolerance of mandarin fruit to CI, and that changes in the expression of genes during storage appear to be
cold-regulated responses, rather than involved in CI tolerance.

1. Introduction functionality (Sevillano et al., 2009; Lafuente et al., 2017). Furthermore,

the stability and functionality of membranes is affected by oxidative
A recurring problem during the commercialization of citrus fruit is processes caused by a cold-induced overproduction and accumulation of
the appearance of peel damage due to the exposure to low temperatures ROS (Suzuki et al., 2012). Experimental evidence indicates that CI in
during postharvest transport and storage, which depreciates their Citrus fruit is associated with a boost in oxidative stress processes,
commercial value. These damages and blemishes caused by low non- exemplified by an enhancement of the expression of genes and enzy­
freezing temperatures are referred to as chilling injury (CI), which is matic activities of the antioxidant system, and a higher singlet oxygen
usually manifested as small depressions in the flavedo that expand, sink quenching capacity (Lado et al., 2016; Lafuente et al., 2017).
and darken with the continuous exposure to cold (Lado et al., 2019a; Accumulation of bioactive compounds with antioxidant properties,
Zacarias et al., 2020). Although mandarin fruit is considered more might play an important role in the tolerance of fruit to CI. The peel of
resistant to CI than fruit of other Citrus species, contrasting predisposi­ citrus fruit contains compounds such as carotenoids, vitamin C and
tion to CI can be observed among different cultivars (Sala, 1998; Rey polyphenols which are part of the non-enzymatic defense mechanism
et al., 2020). against oxidative stress in plant tissues (Zou et al., 2016). Carotenoids
Exposure to cold temperatures has a direct impact on the integrity of are potent lipid-soluble antioxidants and have been associated with the
cell membranes (Lyons, 1973), and accelerates changes in membranes tolerance of grapefruit (Lado et al., 2015, 2016) and mandarin to CI (Rey
composition and conformation which negatively affects their et al., 2020). On the other hand, changes in the concentration of vitamin

* Corresponding author.
E-mail address: [email protected] (L. Zacarias).

https://doi.org/10.1016/j.postharvbio.2021.111594
Received 7 April 2021; Received in revised form 13 May 2021; Accepted 13 May 2021
Available online 29 May 2021
0925-5214/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

C in the peel of citrus fruit appears to be a response to low temperatures,

but its involvement in CI tolerance is still controversial (Lado et al.,
2016; Rey et al., 2020). Tocopherols are highly efficient lipophilic an­
tioxidants (Munné-Bosch and Alegre, 2002; Falk and Munné-Bosch,
2010) and have been detected in the flavedo of citrus fruit (Mathaba
et al., 2014; Assefa et al., 2017), but their role in the sensibility/tol­
erance of citrus fruit to CI is currently unknown.
Tocopherols are plant isoprenoids belonging to the chemical family
of tocochromanols, which also includes tocotrienols, tocomonoenols
and plastochromanol 8. All tocochromanols are formed by a polar
chromanol ring and a hydrophobic polyprenyl side chain that varies
according to the specific tocochromanol, and is fully saturated in the
case of tocopherols. In addition, four natural sub-forms of tocopherols
exist according to the degree of methylation and position of the chro­
manol ring: δ-, β-, γ- and α-tocopherol. They participate in a variety of
plant metabolic processes (Munné-Bosch and Alegre, 2002; Falk and
Munné-Bosch, 2010), and tocopherols, together with tocotrienols, are
the only natural compounds that exhibit vitamin E activity in animal
cells, with α-tocopherol being the most potent vitamin E form (Traber
and Sies, 1996).
Tocopherols can scavenge and quench lipid peroxyl radicals and
ROS, such as singlet oxygen, protecting plant tissues from lipid peroxi­
dation and oxidative damage (Sies and Stahl, 1995; Foyer and Noctor,
2005; Havaux et al., 2005; Mène-Saffrané et al., 2010). Increases in
tocopherol content have been detected in plant tissues in response to
different abiotic stresses, such as high light, salinity, drought, nutrient
deficiency and cold (Bergmüller et al., 2003; Collakova and DellaPenna, Fig. 1. Schematic representation of the tocopherol biosynthetic pathway. The
2003a; Havaux et al., 2005; Maeda et al., 2006; Spicher et al., 2016). intermediates and enzymes catalyzing the reactions are: Phytyl-P, phytyl
They seem to play a crucial role in the adaptation of plants to low phosphate; G3P, glyceraldehyde 3-phosphate; IPP, isopentenyl diphosphate;
temperatures, and mutants impaired in tocopherol synthesis have shown DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl diphosphate; PPP,
a cold-sensitive phenotype, with retarded growth, early senescence and phytyl pyrophosphate; L-tyr, amino acid L-tyrosine; HPP, 4-hydroxyphenylpyr­
reduced seed germination under cold stress (Havaux et al., 2005; Maeda uvate; HGA, homogentisate; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol;
et al., 2006; Wang et al., 2017). However, the involvement of tocopherol DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinol; VTE5, phytol kinase; VTE6,
metabolism in the response of fruit to cold stress during postharvest phytyl-P kinase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; GGPPS, GGPP
synthase; GGDR, GGPP reductase; TAT, tyrosine aminotransferase; HPPD, HPP
storage has been scarcely studied.
dioxygenase; VTE2, homogentisate phytyltransferase (HPT); VTE3, MPBQ
Although the tocopherol biosynthetic pathway was elucidated
methyltransferase (MPBQ-MT); VTE1, tocopherol cyclase (TC); VTE4, tocoph­
several years ago, key genes encoding enzymes of the main biosynthetic
erol methyltransferase (γ-TMT).
steps have been recently identified (Fritsche et al., 2017; Mène-Saffrané,
2017; Pellaud and Mène-Saffrané, 2017; Muñoz and Munné-Bosch,
aminotransferases (TATs), and the loss of function of TAT1 in Arabi­
2019). The first committed step of the tocopherol-core pathway is the
dopsis resulted in a reduction of TAT activity and tocopherol levels in
condensation of homogentisate (HGA) with phytyl pyrophosphate (PPP)
leaves (Riewe et al., 2012). The synthesis of HGA from HPP is catalyzed
(Fig. 1). While HGA is the common precursor of all tocochromanols, PPP
by HPP dioxygenase (HPPD), and null HPPD Arabidopsis mutants were
is the specific polyprenyl donor substrate for tocopherols. This reaction
deficient in tocopherols (Norris et al., 1998), while its overexpression
is catalyzed by homogentisate phytyl transferase (HPT), encoded by the
only generated a modest increase of total tocopherols in leaves and seeds
VTE2 gene, and results in the formation of 2-methyl-6-phytyl-1,4-benzo­
of Arabidopsis and other species (Tsegaye et al., 2002; Karunanandaa
quinol (MPBQ) (Collakova and DellaPenna, 2003b). Later, MPBQ can
et al., 2005).
either be directly converted into δ-tocopherol first and then into
The specific polyprenyl substrate for tocopherol synthesis PPP can
β-tocopherol by tocopherol cyclase (TC, encoded by VTE1) and
either be derived from geranylgeranyl diphosphate (GGPP) (MEP
γ-tocopherol methyltransferase (γ-TMT, encoded by VTE4); or, it can be
pathway) or from free phytol (formed during chlorophyll degradation).
first methylated by MPBQ methyltransferase (MPBQ-MT, encoded by
Several studies have suggested that the majority of PPP used for
VTE3) resulting in 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ).
tocopherol synthesis comes from the recycling of phytol (Karunanandaa
DMPBQ can be subsequently cyclized into γ-tocopherol and later
et al., 2005; Valentin et al., 2006). Phytol is phosphorylated to phytyl
methylated into α-tocopherol by the same TC and γ-TMT mentioned
phosphate by a phytol kinase (VTE5), and later forms PPP by a phytyl
before. The activity of HPT, and expression of VTE2, has been proven to
phosphate kinase (VTE6). In leaves and seeds of Arabidopsis, it has been
limit tocopherol synthesis in leaves and seeds of Arabidopsis thaliana
estimated that most of the PPP used for tocopherol synthesis is derived
(Savidge et al., 2002; Collakova and DellaPenna, 2003b), while
from the VTE5-dependent recycling of phytol (Valentin et al., 2006).
expression of VTE3 and VTE4 appears to shape tocopherol composition
Similar results have been observed in fruit and leaves of tomato, where
rather than affecting their content (Bergmüller et al., 2003; Cheng et al.,
the down-regulation of VTE5 strongly impaired tocopherol production
2003; Collakova and DellaPenna, 2003b).
(Almeida et al., 2016). Changes in the expression of VTE6 also have a
Tocopherol precursors HGA and PPP are formed in independent
direct impact in tocopherol accumulation (vom Dorp et al., 2015),
metabolic pathways and their availability have a strong impact in the
indicating that the supply of PPP through the recycling of phytol formed
final amount of tocopherol accumulated in a tissue (Karunanandaa et al.,
during chlorophyll degradation has an important participation in the
2005). The precursor HGA, which confers the polar chromanol ring,
synthesis of tocopherols in plants. Moreover, genes of the MEP pathway,
derives from 4-hydroxyphenylpyruvate (HPP) formed by the degrada­
such as DXS, also influence tocopherol concentrations in plant tissues
tion of the amino acid L-tyrosine (L-tyr) produced by the shikimate (SK)
(Estévez et al., 2001; Rodríguez-Concepción and Boronat, 2015).
pathway. The conversion of L-tyr into HPP is catalyzed by tyrosine

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

In Citrus, information about tocopherol content and biosynthesis is (Teknokroma, Barcelona, Spain), maintained at room temperature, and
very limited and only a few studies have assessed their accumulation in a ternary gradient elution (Table S1) with methanol, methyl tert-butyl
different tissues. Tocopherols have been detected in the peel of orange, ether and water, at a flow rate of 1 mL min− 1. Compounds were
lemon and other less known cultivars, mainly in the form of α- and detected by fluorescence with excitation and emission wavelength at
γ-tocopherol, and content and proportion of each form varies depending 296 and 340 nm, respectively. Identification and quantification of the
on the specie (Mathaba et al., 2014; Assefa et al., 2017). However, the different tocopherols was achieved by comparison with the retention
identification of the main tocopherol biosynthetic genes, and how they times and peak areas of authentic standards of δ-, γ- and α-tocopherol
are regulated in citrus fruit in response to abiotic stress conditions is (Sigma-Aldrich, Barcelona, Spain). All procedures were carried out on
currently unknown. Since tocopherols are widely recognized by their ice and under dim light to prevent degradation. Total tocopherol content
antioxidant capacity, our working hypothesis envisages the potential was calculated as the sum of the different tocopherol isoforms. Con­
participation of these compounds in the responses of citrus fruit to centrations are expressed as mg kg− 1 of fresh weight. Samples were
postharvest cold storage. To unravel this objective, tocopherol content extracted twice and results are the mean of two replicates (mean ±
and expression of 14 genes involved in tocopherol biosynthesis were standard deviation).
analyzed in the peel of fruit of three mandarin cultivars differing in their
sensitivity to CI (‘Fortune’ >‘Nova’ > ‘Nadorcott’) stored at 2 ◦ C for up 2.4. Identification of the genes involved in tocopherol biosynthesis in
to 8 weeks. Citrus and primers design

2. Materials and methods In order to identify Citrus genomic sequences for the main enzymatic
steps involved in the tocopherol biosynthesis pathway, and in HGA and
2.1. Plant material and storage conditions PPP synthesis, peptide sequences of previously described genes (Fig. 1)
from Arabidopsis thaliana and Solanum lycopersicum were obtained from
Fruit of three mandarin cultivars with contrasting susceptibility to the TAIR10 and iTAG2.4 databases (Table 1). These Arabidopsis and
develop chilling symptoms during postharvest cold stored were selected: tomato protein sequences were used to perform a TBLASTN search
‘Fortune’ (Citrus clementina Hort. Ex Tanaka x Citrus reticulata Blanco), against the Citrus sinensis (C. sinensis) genome databases Phytozome (JGI
high sensitivity; ‘Nova’ (Citrus clementina Hort. ex Tanaka x (Citrus Phytozome v12; Wu et al., 2014) and Huazhong Agricultural University
paradisi McFadyen x Citrus reticulata Blanco)), medium sensitivity; and (http://citrus.hzau.edu.cn/orange; Xu et al., 2013). Protein sequence
‘Nadorcott’ (Citrus reticulate, Blanco), low sensitivity (Rey et al., 2020). identity between the closest orthologous genes was performed using the
Fruit were harvested at commercial maturity from adult trees growing DNAman software (Lynnon Biosoft, Quebec, Canada), and genes were
under standard agronomical conditions, located in a commercial or­ selected based on the percentage of identity at the amino acid level
chard (Lliria, Valencia, Spain) or in The Citrus Germplasm Bank at the (Table 1). A BLAST search against the C. sinensis genome with the
Instituto Valenciano de Investigaciones Agrarias (IVIA, Moncada, nucleotide sequences of the selected C. sinensis genes was performed to
Valencia, Spain). Immediately after harvest, fruit were delivered to the corroborate homology within the genome that might interfere in primer
laboratory, inspected for size uniformity, and fruit free of visible peel design. Genes with high similarity were taken into account in the design
damage and defects were stored at 2 ◦ C and 80–85 % RH for up to 8 of primers to assure specificity. Primer pairs were design manually or
weeks. At harvest and after 1, 3, 5 and 8 weeks of cold storage, flavedo using the online software PCR Efficiency calculator (http://130.60.24
tissue (colored part of the rind) was excised with a scalpel, frozen in .89/efficiency.html), and specificity was corroborated by sequencing
liquid nitrogen, ground to a fine powder and stored at − 80 ◦ C until the PCR products.
analysis.
2.5. RNA extraction and cDNA synthesis
2.2. Chilling injury evaluation
Total RNA was isolated from flavedo tissue using the RNeasy Plant
Mandarin fruit of each cultivar was visually inspected for CI symp­ Mini Kit (Qiagen, Madrid, Spain). Total RNA was treated with DNase I
toms during the storage period. Fruit were scored in a scale from 0 to 3, (DNA free, DNase treatment & removal, Ambion, Barcelona, Spain), and
according to the intensity and extension of the symptoms, as previously RNA concentration and quality was measured by spectrophotometric
described (Rey et al., 2020). Results are expressed as the percentage of analysis (Nanodrop, Thermo Fisher Scientific, Barcelona, Spain). After
fruit within each CI category. Three replicate samples of 20 fruit each quantification, the quality of the RNA was verified by 1 % agarose gel
were used for CI evaluation. electrophoresis with GoodView™ Nucleic Acid Stain (SBS Genetech,
Beijing, China). For cDNA synthesis, 5 μg of total RNA were reverse-
2.3. Tocopherol extraction, identification and quantification transcribed using the SuperScript III Reverse Transcriptase (Invi­
trogen, Barcelona, Spain) in a total volume of 20 μL, following the
Tocopherols in the flavedo were extracted following the procedure manufacturer’s procedure. First-strand cDNA samples were diluted 1:10
described by Fraser et al. (2000) with some modifications. A sample of to a final concentration of approximately 100 ng μL− 1 for each ampli­
200 mg of flavedo was extracted with 2 mL of methanol (HPLC grade, fication reaction.
Scharlau, Barcelona, Spain), 1 mL of Tris buffer (50 mM Tris pH 7.5 with
1 M NaCl) and 4 mL of dichloromethane (HPLC grade, Scharlau, Bar­ 2.6. Gene expression analysis by quantitative real time PCR
celona, Spain) with a mortar and pestle with sea sand as an abrasive.
After vortex-mixing, samples were sonicated for 5 min and then Quantitative real-time PCR was performed on a LightCycler 480 in­
centrifuged for 10 min at 3000 g and 4 ◦ C. After this, the dichloro­ strument (Roche, Madrid, Spain), using the LightCycler 480 SYBRGreen
methane phase was recovered and the methanol phase and flavedo I Master kit (Roche, Madrid, Spain) and following the manufacturer’s
pellet were re-extracted with 2 mL of dichloromethane. Extracts were instructions. Primer pairs sequences of the genes related to tocopherols
dried under a nitrogen stream and re-suspended in ethyl acetate (HPLC biosynthesis used for the amplification of each gene are listed in
grade, Merck, Madrid, Spain) for chromatography analyses. Tocopherol Table S2. The cycling protocol, for all genes analyzed, consisted of 10
content was determined using a Waters HPLC system (Acquity® Arc™, min at 95 ◦ C for pre-incubation, 40 cycles of 10 s at 95 ◦ C for denatur­
Waters, Barcelona, Spain) coupled with a fluorescence detector (2475 ation, 10 s at 59 ◦ C for annealing and 10 s at 72 ◦ C for extension.
FLR Detector, Waters, Barcelona, Spain). Separation of tocopherols was Fluorescent intensity data was acquired during the extension time.
carried out with a YMC C30 column (150 × 4.6 mm, 3 μm) Specificity of the PCR reaction was assessed by the presence of a single

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

Table 1
Identification of homologous genes and enzymes involved in tocopherol synthesis in Citrus sinensis genome.
c d
Gene Enzyme C. sinensis Protein length (aa) A. thaliana % Id Ath e S. lycopersicum f
% Id Slyc g

a
DXS1 1-deoxy-D-xylulose-5-phosphate synthase orange1.1g003948 784 71 Solyc11g010850 81
AT4G15560
DXS2 1-deoxy-D-xylulose-5-phosphate synthase orange1.1g004923 a 723 82 Solyc01g067890 86
GGPPS1 Geranylgeranyl pyrophosphate synthase orange1.1g019903 a 334 AT4G38460 73 Solyc09g008920 78
GGPPS6 Geranylgeranyl pyrophosphate synthase orange1.1g037716 a 354 AT4G36810 59 Solyc11g011240 72
GGDR Geranylgeranyl diphosphate reductase orange1.1g012488 a 462 AT1G74470 84 Solyc03g115980 83
VTE5 Phytol kinase orange1.1g022218a 301 AT5G04490 69 Solyc03g071720 62
VTE6 Phytol-P kinase orange1.1g020374 a 327 AT1G78620 73 Solyc07g062180 84
TAT1 Tyrosine aminotransferase orange1.1g014936 a 415 AT5G53970 75 Solyc10g007110 72
HPPD 4-hydroxyphenylpyruvate dioxygenase orange1.1g013898 a 434 AT1G06570 77 Solyc07g045050 78
VTE2 Homogentisate phytyl transferase (HPT) Cs4g03670.1 b 411 AT2G18950 68 Solyc07g017770 65
VTE3a Dimethyl-phytylquinol methyl transferase orange1.1g019479 a 340 87 Solyc03g005230 88
AT3G63410
VTE3b Dimethyl-phytylquinol methyl transferase orange1.1g019684 a 337 76 Solyc09g065730 90
VTE1 Tocopherol cyclase (TC) orange 1.1g011258 a 490 AT4G32770 68 Solyc08g068570 76
VTE4 γ-tocopherol methyl transferase (γ-TMT) orange 1.1g016921 a 380 AT1G64970 67 Solyc08g076360 72
a
Phytozome v12.1, Citrus sinensis v1.1 (Sweet orange) genome database.
b
Huazhong Agricultural University, Citrus sinensis (Sweet orange) genome database.
c
Predicted protein size in Phytozome v12.1 (aa, amino acids).
d
TAIR10, Arabidopsis thaliana locus.
e
% Id Ath: percentage of identity with the corresponding Arabidopsis thaliana protein sequence.
f
Phytozome v12.1, Solanum lycopersicum iTAG2.4 (Tomato) genome database.
g
% Id S.lyc: percentage of identity with the corresponding Solanum lycopersicum protein sequence.

peak in the dissociation curve performed after the amplification steps. 3. Results
For expression measurements, we used the LightCycler 480 Software
release 1.5.0, version 1.5.0.39 (Roche, Madrid, Spain) and calculated 3.1. Differences in the susceptibility to chilling injury of mandarin fruit
expression levels relative to values of a reference sample using the during cold storage
Relative Expression Software Tool (Pfaffl et al., 2002). The house­
keeping gene used for normalization was ACTIN (Alós et al., 2014). For To investigate the role of tocopherols in the susceptibility/tolerance
all cultivars and dates analyzed, the reference sample used to calculate of citrus fruit to CI during postharvest cold storage, we selected fruit of
relative expression was the value of each gene in the flavedo of ‘Fortune’ three mandarin cultivars with contrasting differences in their suscepti­
fruit at harvest (time 0), which was set at 1. bility to CI (Rey et al., 2020). ‘Fortune’ mandarin fruit were very prone
to develop CI and, after 3 weeks of storage at 2 ◦ C, CI affected almost 90
2.7. Statistical and principal components analysis % of the fruit (40 % of fruit had severe symptoms). The severity of CI
increased with storage time and, after 5 and 8 weeks of storage, 62 %
To determine if mean differences were significant among storage and 90 % of the fruit, respectively, developed severe CI (Fig. 2). Fruit of
dates in each mandarin cultivar, data for γ- and α-tocopherol content ‘Nova’ mandarin developed CI symptoms more slowly than those of
was subjected to a one-way analysis of variance (ANOVA) followed by ‘Fortune’ and the proportion of fruit with high and severe damage was
Tukey’s test (significance level at p ≤ 0.05), using the InfoStat software 48 % and 74 % after 5 and 8 weeks of storage, respectively. By contrast,
(version 2018, Grupo InfoStat, Córdoba, Argentina). Principal compo­ fruit of ‘Nadorcott’ proved to be highly resistant to CI. After 5 weeks at 2
nent analysis (PCA) was built using data of tocopherol content and ◦
C only 6 % of fruit developed slight symptoms, and by week 8, 74 % of
relative gene expression at harvest and at the four storage dates analyzed the fruit was still healthy (Fig. 2).
for the three mandarin cultivars, using the packages ‘FactoMineR’
(version 2.4) and ‘factoextra’ (version 1.0.7) in RStudio (version
3.2. Tocopherol content in the flavedo of mandarin fruit during cold
1.3.1093, RStudio Team, PBC, Boston, MA, United States).
storage

Accumulation of tocopherols was determined in the flavedo of fruit

Fig. 2. Chilling injury (CI) incidence (% of fruit) according to severity of symptoms in fruit of ‘Fortune’, ‘Nova’ and ‘Nadorcott’ mandarins stored at 2 ◦ C for 3, 5 and
8 weeks.

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

of the three mandarin cultivars during storage at 2 ◦ C (Fig. 3). Of the geranylgeranyl diphosphate reductase (GGDR); 2 genes involved in
four naturally occurring tocopherol forms (α, β, δ and γ), only α- and phytol catabolism, phytol kinase (VTE5) and phytol-P kinase (VTE6); 2
γ-tocopherol were detected in the flavedo of mandarin fruit. In all the genes of the SK pathway, tyrosine aminotransferase (TAT1) and
samples analyzed, α-tocopherol was the predominant form, accounting 4-hydroxyphenylpyruvate dioxygenase (HPPD); and 5 genes specifically
for 78.4 %, 86.0 % and 94.7 %, on average, of total tocopherols in involved in the tocopherol core-pathway: homogentisate phytyl trans­
‘Nova’, ‘Nadorcott’ and ‘Fortune’, respectively. ferase (VTE2), 2 isoforms of 2-methyl-6-phytyl-benzoquinol methyl
Total tocopherols at harvest, calculated as the sum of γ- and transferase (VTE3a and VTE3b), tocopherol cyclase (VTE1) and
α-tocopherol, were higher in the flavedo of ‘Nadorcott’ (135 mg kg− 1) γ-tocopherol methyl transferase (VTE4). Protein length and percentage
than in ‘Nova’ (90 mg kg− 1) and ‘Fortune’ (54 mg kg− 1) (Fig. 3). These of identity of the sequences selected with the corresponding Arabidopsis
differences in total tocopherols among cultivars were mainly due to the thaliana and Solanum lycopersicum orthologous are summarized in
low accumulation of α-tocopherol in ‘Nova’ and ‘Fortune’ (40 and 60 % Table 1. Identity of the protein sequences ranged between 59 and 87 %
lower, respectively) in comparison with ‘Nadorcott’ mandarin. The in comparison with the corresponding sequences of Arabidopsis and
concentration of γ-tocopherol was similar between ‘Nadorcott’ and between 62 and 90 % with those of tomato (Table 1).
‘Nova’ mandarins, and about 4-times higher than that detected in
‘Fortune’. During storage at 2 ◦ C, total tocopherols and α-tocopherol
remained constant in ‘Fortune’ and ‘Nova’, while in ‘Nadorcott’ they 3.4. Comparison of the expression of genes involved in tocopherol
experienced an initial reduction after 1 week of storage, increasing biosynthesis in the flavedo of mandarin fruit during cold storage
gradually to reach concentrations similar to the initial levels at the end
of the storage (Fig. 3). The concentration of γ-tocopherol during cold The transcriptional profiling of 14 genes involved in tocopherol
storage remained constant in fruit of ‘Fortune’, but significantly synthesis (Table 1) was analyzed in the flavedo of ‘Fortune’, ‘Nova’ and
increased in ‘Nova’ (74.5 %) and in ‘Nadorcott’ (55.1 %) mandarins. ‘Nadorcott’ mandarin stored for up to 8 weeks at 2 ◦ C, in order to un­
Despite these fluctuations, the differences in tocopherol concentrations derstand whether the expression patterns of these genes are related to
among mandarin cultivars were maintained during the whole cold the differences in tocopherol content and CI susceptibility. Figs. 4 and 5
storage period (Fig. 3). show the changes in the expression of genes of the chlorophyll degra­
dation and MEP pathways, respectively, involved in the formation of the
metabolic precursors PPP (Fig. 1). In the flavedo of fruit at harvest, when
3.3. Selection of genes involved in tocopherol biosynthesis in Citrus the differences in total tocopherols among cultivars were already
evident, major differences were found in genes of the MEP pathway, but
With the aim of understanding the transcriptional regulation of not in those involved in the recycling of phytol. Accumulation of VTE5
tocopherol biosynthesis during postharvest cold storage of citrus fruit, a and VTE6 transcripts were similar in fruit of the three cultivars at harvest
search of genes corresponding to key enzymatic steps of the different and increased with the storage period. In general, these transcripts
pathways involved in tocopherol production was performed in the Citrus accumulated to lower levels in the flavedo of the high-accumulating
sinensis genome, based on genes previously identified in Arabidopsis tocopherol cultivar ‘Nadorcott’, than in ‘Nova’ or ‘Fortune’ mandarin
thaliana and Solanum lycopersicum (Table 1). Tocopherol accumulation (Fig. 4).
in plant tissues has been shown to be dependent not only on the tran­ With the exception of GGPPS1, whose mRNA was lower in fruit of
scriptional changes of specific genes of the tocopherol core-pathway, but ‘Nadorcott’ at harvest than in the other two cultivars, expression of other
also of the genes involved in the availability of the precursors PPP and genes of the MEP pathway analyzed (DXS1, DXS2, GGPPS6 and GGDR)
HGA (Mène-Saffrané, 2017; Muñoz and Munné-Bosch, 2019). HGA is were between 2 and 3-times higher in the flavedo of ‘Nova’ and
formed via the SK pathway, whereas PPP can be formed de novo from ‘Nadorcott’ than in ‘Fortune’ mandarin (Fig. 5). Cold storage up-
GGPP (MEP pathway) or by the recycling of free phytol (formed during regulated the expression of most genes (except GGDR), although dif­
chlorophyll degradation) (Pellaud and Mène-Saffrané, 2017). Therefore, ferences in the timing of induction were found among cultivars (Fig. 5).
a total of 14 genes were selected for further expression analysis (Fig. 1), In general, accumulation of most transcripts after 8 weeks of storage
corresponding to 5 genes of the MEP pathway: 2 isoforms of 1-deoxy- were higher than at harvest. Moreover, GGDR was the only gene dis­
D-xylulose-5-phosphate synthase (DXS1 and DXS2), 2 isoforms of ger­ playing a contrasting expression pattern in ‘Fortune’ mandarin than in
anylgeranyl pyrophosphate synthase (GGPPS1 and GGPPS6) and ‘Nova’ and ‘Nadorcott’, with a progressive up-regulation in the former

Fig. 3. Total tocopherols content (mg kg− 1, as the sum of α- and γ-tocopherol) in the flavedo of ‘Fortune’, ‘Nova’ and ‘Nadorcott’ mandarins stored at 2 ◦ C for up to 8
weeks. Capital letters indicate significant differences in α-tocopherol, while lowercase letters indicate significant differences in γ-tocopherol content among storage
dates for each cultivar (p ≤ 0.05, Tukey test).

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

Fig. 4. Relative expression of the genes of the chlorophyll degradation pathway

involved in the synthesis of PPP (phytyl pyrophosphate) in the flavedo of
‘Fortune’, ‘Nova’ and ‘Nadorcott’ mandarins stored at 2 ◦ C for up to 8 weeks.
The genes analyzed were VTE5 (phytol kinase) and VTE6 (phytyl-P kinase). An
expression value of 1 was arbitrarily assigned to the values obtained in the
flavedo of ‘Fortune’ fruit at harvest. The data are mean ± S.E of at least
three replicates.

and a decline in the peel of the other two mandarins (Fig. 5).
The expression pattern of the two genes belonging to the SK pathway
analyzed, TAT1 and HPPD, also showed differences among cultivars
(Fig. 6). At harvest, accumulation of both transcripts was slightly higher
in the peel of ‘Nadorcott’ and ‘Nova’ than in ‘Fortune’. Cold storage
produced a transient reduction in the accumulation of TAT1 transcript,
which was recovered after 5 or 8 weeks of storage. By contrast, HPPD
gene was markedly up-regulated during cold storage, reaching levels 4-,
2- and 6.5-times higher after 5 weeks than initial ones in the peel of
‘Fortune’, ‘Nova’ and ‘Nadorcott’, respectively. Transcripts levels
declined after 8 weeks of storage in ‘Nova’ and ‘Nadorcott’ (Fig. 6).
Genes of the specific tocopherol biosynthetic pathway also exhibited
differences in their expression pattern among cultivars at harvest and
after cold storage (Fig. 7). At harvest, expression of these genes tended to
be higher in the flavedo of ‘Nadorcott’ and ‘Nova’, which accumulated
higher tocopherol content. Expression of VTE3a was 4- and 5-times
higher in ‘Nadorcott’ and ‘Nova’, respectively, than in ‘Fortune’, Fig. 5. Relative expression of genes of the MEP pathway involved in the syn­
whereas VTE4 was about 30 % higher in these two cultivars. By contrast, thesis of PPP (phytyl pyrophosphate) in the flavedo of ‘Fortune’, ‘Nova’ and
transcripts corresponding to VTE3b and VTE1 were higher in ‘Nadorcott’ ‘Nadorcott’ mandarins stored at 2 ◦ C for up to 8 weeks. The genes analyzed
than in ‘Nova’ and ‘Fortune’, and VTE2 was the only gene with higher were DXS1 and DXS2 (1-deoxy-D-xylulose-5-phosphate synthase 1 and 2),
expression levels in ‘Nova’ than in the other cultivars (Fig. 7). Analysis of GGPPS1 and GGPPS6 (geranylgeranyl pyrophosphate synthase 1 and 6) and
the expression of these genes during storage at 2 ◦ C revealed two pat­ GGDR (geranylgeranyl diphosphate reductase). An expression value of 1 was
terns: a down-regulation for VTE3b and VTE4, and an initial decline only arbitrarily assigned to the values obtained in the flavedo of ‘Fortune’ fruit at
harvest. The data are mean ± S.E of at least three replicates.
to increase again at the end of the storage for VTE2, VTE3a and VTE1.
After 8 week of storage, relevant differences among cultivars were
detected for VTE2 and VTE3a, with expression levels being highest in 3.5. Multivariate analysis of tocopherol accumulation and changes in
‘Nova’ mandarin (Fig. 7). gene expression in the flavedo of mandarin fruit during cold storage
A global comparison of the changes in gene expression among cul­
tivars revealed that the relative expression of the majority of genes (with A principal component analysis (PCA) was carried out to explore the
the exception of GGPPS1 and VTE2) involved in the different tocopherol relationship between tocopherol accumulation and gene expression in
synthetic pathways were always lower in the peel of ‘Fortune’ (low response to cold storage among the different cultivars. The first three
tocopherol content) than in the other cultivars with high tocopherol principal components (PCs) explained for 70 % of the total variance of
content. the data (PC1 = 26.4 %, PC2 = 25.1 % and PC3 = 17.8 %). The score and
variable plots for PC1 vs PC2 (Fig. 8A and B) and PC1 vs PC3 (Fig. 8C and
D) are presented. The two first PC tended to separate samples according
to the storage dates rather than the different cultivars (Fig. 8A). Variable
plot (Fig. 8B) revealed that the expression of genes VTE3a, VTE3b, VTE4

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

Fig. 6. Relative expression of genes of the SK pathway involved in the synthesis

of HGA (homogentisate) in the flavedo of ‘Fortune’, ‘Nova’ and ‘Nadorcott’
mandarins stored at 2 ◦ C for up to 8 weeks. The genes analyzed were TAT1
(tyrosine aminotransferase) and HPPD (4-hydroxyphenylpyruvate dioxyge­
nase). An expression value of 1 was arbitrarily assigned to the values obtained
in the flavedo of ‘Fortune’ fruit at harvest. The data are mean ± S.E of at least
three replicates.

and TAT1 positively contributed to PC1, while HPPD and DXS2

contributed negatively. The first genes tended to be repressed by cold
and their relative expression was highest at harvest (Fig. 8A and B),
while HPPD and DXS2 exhibited a sharp induction by cold and were
highest during mid-storage (3 w). Variables contributing to PC2 were
mainly GGPPS1, GGPPS6, VTE2, VTE6 and Gtoc (γ-tocopherol). These
variables were grouped together and were genes up-regulated (gradual
or sharply) towards the end of cold storage. The high correlation of
γ-tocopherol with GGPPS1, GGPPS6, VTE6 and VTE2 suggests that the
induction of these genes could explain the increase detected in
γ-tocopherol content in ‘Nova’ and ‘Nadorcott’ at the end of the storage.
When the PC3 was plotted against PC1 (Fig. 8C), ‘Nadorcott’ differen­
tiated from the other two cultivars, which grouped together. The main
variables contributing to PC3 were Ttoc (total tocopherols) and Atoc
(α-tocopherol), and to a lesser extent the expression of VTE1, high­
lighting the differences in total and α-tocopherol content between
‘Nadorcott’ and the other cultivars. Moreover, the poor representation of Fig. 7. Relative expression of genes of the tocopherol-core pathway in the
GGDR in the selected PCs is likely due to the contrasting tendency of its flavedo of ‘Fortune’, ‘Nova’ and ‘Nadorcott’ mandarins stored at 2 ◦ C for up to 8
expression in response to cold among the cultivars. weeks. The genes analyzed were VTE2 (HPT, homogentisate phytyl transferase),
VTE3a and VTE3b (MPBQ-MT, 2-methyl-6-phytyl-1,4-benzoquinol methyl­
4. Discussion transferase a and b), VTE1 (TC, tocopherol cyclase) and VTE4 (γ-TMT,
γ-tocopherol methyltransferase). An expression value of 1 was arbitrarily
assigned to the values obtained in the flavedo of ‘Fortune’ fruit at harvest. The
Tocopherols are plastid-derived isoprenoids that play important
data are mean ± S.E of at least three replicates.
physiological functions in plants, of which their role as antioxidants is
one of the most relevant (Falk and Munné-Bosch, 2010; Fritsche et al.,
2017; Muñoz and Munné-Bosch, 2019). Tocopherols can either scavenge fruit to CI during storage at 2 ◦ C. To that end, we analyzed changes in
peroxyl radicals or quench singlet oxygen, being highly efficient in tocopherol content and in the expression of 14 genes involved in the
protecting plant tissues against lipid peroxidation and oxidative damage synthesis of tocopherol precursors and in the specific steps shaping to­
(Falk and Munné-Bosch, 2010). Experimental evidences indicate that CI copherols composition in the peel of mandarin fruit with contrasting
development in Citrus fruit is an event associated with a boost in susceptibility to CI. ‘Fortune’ mandarin was highly sensitive to CI,
oxidative processes (Sala, 1998; Lado et al., 2016), and that the selective whereas ‘Nova’ and ‘Nadorcott’ mandarins displayed moderated and
accumulation of certain antioxidant compounds, such as specific ca­ low CI, respectively (Fig. 2). To our knowledge, this is first physiological
rotenoids, may be involved in the tolerance to this type of stress (Lado and molecular characterization of tocopherols biosynthesis in Citrus fruit
et al., 2015, 2016; Rey et al., 2020). To gain further insights into the in an attempt to understand their involvement in the postharvest
implication of other antioxidants in the susceptibility of Citrus fruit to CI, tolerance to cold stress.
in this work we have focused on the involvement of tocopherols, as Analysis of tocopherol content revealed that only the forms α- and
highly efficient lipid-soluble antioxidants, in the tolerance of mandarin γ-tocopherol accumulated in the flavedo of the three mandarin cultivars
at harvest and during cold storage (Fig. 3). Moreover, α-tocopherol was

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

Fig. 8. Principal components analysis (PCA) score plot (A) and variables plot (B) for PC1 vs PC2, and score plot (C) and variables plot (D) for PC1 vs PC3 of the
variables analyzed (tocopherol content and relative gene expression) in ‘Fortune’ (F), ‘Nova’ (N) and ‘Nadorcott’ (ND) mandarin. Variables are colored in a gradient
from blue to orange according to the square cosine (cos2), which reflects the representation quality of a variable in the selected PC axes (blue: low cos2, bad
representation; orange: high cos2, good representation). Total tocopherol (Ttoc), α-tocopherol (Atoc) and γ-tocopherol (Gtoc) content are highlighted in circles.

the main form, accounting for 92.8 %, 82.3 % and 87.8 % of total to­ harvest revealed marked differences among cultivars. ‘Nadorcott’
copherols at harvest in ‘Fortune’, ‘Nova’ and ‘Nadorcott’ mandarin, accumulated the highest tocopherol content (135 mg kg− 1), which was
respectively, and these proportions remained relatively stable or slightly about 1.5- and 2.5-times higher than that of ‘Nova’ and ‘Fortune’,
decreased during 2 months of storage at 2 ◦ C (Fig. 3). Information respectively (Fig. 3). These concentrations of total tocopherols were
related to tocopherol accumulation in citrus fruit is limited, but in fruit higher than those previously reported by Assefa et al. (2017) in fruit of
of other Citrus species, α- and γ-tocopherol have been the detected forms other Citrus species (70–131 mg kg− 1 of dry weight). As with other
(Mathaba et al., 2014; Assefa et al., 2017). In orange and tangerine bioactive molecules (Alquezar et al., 2008; Alós et al., 2014; Assefa
grown in Korea, α-tocopherol represented 40 % and 36 % of total to­ et al., 2017), tocopherol contents in the peel were much higher to those
copherols, respectively (Assefa et al., 2017), which is significantly lower reported in the pulp (Chun et al., 2006). Interestingly, we have recently
than those determined in this work. If these discrepancies represent reported that the concentration of total carotenoids in the flavedo of
genotypic, environmental or growing conditions differences remain to ‘Nadorcott’ mandarin is substantially higher than that of ‘Fortune’ and
be determined. Composition of tocopherols varies among different plant ‘Nova’ (Rey et al., 2020), and also of other traditional mandarin (Lado
species but, in general, α-tocopherol is the major form in most plant et al., 2019b), suggesting an enhanced synthesis and accumulation of
tissues, with the exception of seeds of certain species which accumulate isoprenoids-derived compounds in fruit of this mandarin cultivar.
mainly γ-tocopherol (Munné-Bosch and Alegre, 2002; Horvath et al., Comparison of tocopherol concentration at harvest and during cold
2006). In fleshly fruit, such as tomato (Quadrana et al., 2013), red storage (Fig. 3) in the peel of the three mandarin cultivars revealed an
pepper (Koch et al., 2002), avocado (Vincent et al., 2020), cherry (Tijero inverse relationship between tocopherol content and the susceptibility
et al., 2016) or olive (Georgiadou et al., 2019), α-tocopherol was also the to CI (Fig. 2). ‘Fortune’ mandarin fruit were extremely susceptible to CI
major form accumulated. and after 3 weeks at 2 ◦ C, 90 % of the fruit exhibited chilling symptoms
Analysis of total tocopherol content in the peel of mandarin at (40 % severe symptoms). At the same storage time, 40 % of ‘Nova’ fruit

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

developed CI (3 % severe) while no damage was detected in ‘Nadorcott’ phytol formed in the degradation of chlorophylls. The expression anal­
(Fig. 3). Since total tocopherols were 60 % and 30 % lower in the ysis of the genes involved in the SK pathway (TAT1 and HPPD) and the
chilling-sensitive fruit of ‘Fortune’ and ‘Nova’, respectively, than in the recycling of phytol (VTE5 and VTE6) (Fig. 1) did not reveal important
CI-tolerant ‘Nadorcott’, it is tempting to speculate that high levels of differences among mandarin genotypes nor with the differences in
tocopherols at the beginning of cold storage may play a protective role tocopherol content (Figs. 4 and 6), suggesting that the expression of
against chilling-induced damage. A similar inverse relationship between these genes does not appear to limit tocopherol concentration in the peel
CI sensitivity and tocopherol levels has been observed in the exocarp of of mandarin fruit. By contrast, the expression of most genes of the MEP
cold stored zucchini fruit, in which higher concentrations were detected pathway at harvest, with the exception of GGPPS1, was significantly
in tolerant genotypes (Rodov et al., 2020). These results are in agree­ lower in the low-tocopherol accumulating cultivar (Fig. 5). These dif­
ment with the general concept of tocopherols in plants under stress ferences were remarkable for the expression of DXS1, DXS2 and GGDR,
conditions, establishing that stress-tolerant species contain higher levels which were 2- to 3-times higher in fruit of ‘Nadorcott’ and ‘Nova’ than in
of tocopherols than sensitive species (Munné-Bosch and Alegre, 2002; ‘Fortune’ mandarin. DXS regulates the amount of IPP produced and
Sadiq et al., 2019). Moreover, although both γ- and α-tocopherol were controls the influx into the MEP pathway (Estévez et al., 2001), while
lower in the peel of ‘Fortune’ fruit, differences between ‘Nova’ and GGDR is the enzyme limiting the reduction of GGPP into precursor PPP.
‘Nadorcott’ were mainly in α-tocopherol, as γ-tocopherol contents were Therefore, if a higher expression of DXS and GGDR genes correlate to
very similar. α-tocopherol has been reported as the most efficient singlet higher enzymatic activity, these enzymatic steps supplying the precursor
oxygen quencher tocopherol sub-form (Gruszka et al., 2008), and it is PPP for condensation with HGA may be important key steps regulating
likely that the differences in this form among the mandarin genotypes the concentration of tocopherols in mandarin. In vegetative tissues, a
may play a role in their contrasting tolerance to CI. down-regulation of the DXS gene has been associated with a lower
During cold storage, total tocopherol and α-tocopherol content did production of plants isoprenoids, including tocopherols (Estévez et al.,
not change in fruit of the CI-sensitive mandarins, but declined after the 2001; Rodríguez-Concepción and Boronat, 2015). Moreover, in tomato
first week in the CI-resistant cultivar, only to recover to similar initial fruit a higher expression of both DXS and GGDR was detected in geno­
levels at the end of storage. In contrast, contents of γ-tocopherol types accumulating high concentrations of tocopherols (Almeida et al.,
significantly increased (almost 2-fold) in ‘Nova’ and ‘Nadorcott’ after 8 2011; Quadrana et al., 2013). Nonetheless, differences in the expression
weeks of storage (Fig. 3). The effect of cold stress on tocopherol accu­ of DXS and GGDR do not explain the higher tocopherol content in
mulation has been mainly studied in vegetative tissues of model plants, ‘Nadorcott’ than in ‘Nova’ (Fig. 3), suggesting the occurrence of other
such as Arabidopsis (Bergmüller et al., 2003; Maeda et al., 2006; Gabruk downstream metabolic steps or post-transcriptional mechanisms
et al., 2016; Sadiq et al., 2019), and only a few studies have evaluated (Hemmerlin, 2013) regulating tocopherol concentrations in mandarin
the influence of this abiotic stress on tocopherol synthesis in fruit. In fruit.
avocado, a short cold-shock increased γ- but not α- and β-tocopherol The condensation of PPP with HGA to form MPBQ, catalyzed by HPT
levels, whereas after 10 d under cold conditions α-tocopherol content (VTE2) (Fig. 1), determines the entrance into the tocopherol-core
decreased by a 20 % (Vincent et al., 2020). In cherry fruit, a transient pathway, which after subsequently methylation and cyclization define
increase in total tocopherol levels was detected after 3 d of storage the final content and composition of tocopherols (Fritsche et al., 2017;
(Tijero et al., 2016). The decrease in α- tocopherol after a 1 week of cold Mène-Saffrané, 2017). The expression of these genes, and in particular
storage in ‘Nadorcott’ mandarin is similar to that observed in avocado of VTE3 isoforms, tended to be higher in the peel of fruit accumulating
and may indicate a primary cold-shock response in this tolerant geno­ higher concentration of tocopherols at harvest (‘Nadorcott’ and ‘Nova’)
type, but the potential influence of this change on CI-tolerance remains than in ‘Fortune’ mandarin (Fig. 7). Although the gene VTE2 is a limiting
to be determined. Despite these fluctuations, changes in total tocoph­ step in tocopherol synthesis in leaves and seeds of Arabidopsis, rice and
erols in fruit of mandarin under low temperature storage are similar to soybean, among others (Collakova and DellaPenna, 2003b; Maeda et al.,
those occurring in other antioxidants compounds, such as ascorbate and 2006; Wang et al., 2017), in mandarin fruit VTE2 did not seem to restrict
glutathione, which experienced minor variations and remained almost tocopherol synthesis since its expression was low in the cultivar with
unchanged during cold storage (Lado et al., 2016; Rey et al., 2020). higher total tocopherols. Our results are in agreement with those of
These results may indicate that the pool of these compounds appears to tomato and olive fruit (Quadrana et al., 2013; Georgiadou et al., 2019),
be metabolically stable during postharvest cold storage or alternatively in which VTE2 appears not to be limiting for tocopherol accumulation.
indicative of a rapid turnover. Therefore, it seems that in the peel of Interestingly, the expression of both isoforms of VTE3 were lower in the
mandarin fruit the concentration of tocopherols, and in particular of peel of ‘Fortune’ (low tocopherol content), suggesting that the expres­
α-tocopherol, at harvest and during the storage period may be an sion of these genes may be relevant for the accumulation of γ- and
important factor for the fruit to cope with postharvest cold stress and in α-tocopherol in mandarin fruit peel. Likewise, a positive correlation
the tolerance to CI development. between transcript levels of VTE3 and tocopherol content and compo­
Tocopherol biosynthesis in plants is very complex and involves the sition was detected in tomato fruit (Quadrana et al., 2013). Moreover,
convergence of different metabolic pathways, which are also connected the last steps of the pathway controlling the final balance between the
and provide intermediates for the synthesis of other relevant compounds different isoforms of tocopherols are catalyzed by TC (VTE1) and γ-TMT
(Mène-Saffrané, 2017; Muñoz and Munné-Bosch, 2019). In this study, (VTE4) (Porfirova et al., 2002; Bergmüller et al., 2003). Expression of
we have analyzed the transcriptional profiling of 14 genes involved in both genes at harvest were slightly higher in fruit of ‘Nadorcott’ man­
the synthesis of the metabolic precursors PPP and HGA, and in the main darin, but differences were minor among cultivars and may not explain
steps of the tocopherol-core pathway, at harvest and during postharvest tocopherol content. Collectively, these results suggest that the differ­
storage of mandarin fruit. The Citrus genes were selected on the basis of ences in tocopherol concentration in the fruit peel of the different
sequence homology with previously identified genes from Arabidopsis mandarin cultivars at harvest appear to be dependent of the upstream
and tomato (Almeida et al., 2011; Mène-Saffrané, 2017; Muñoz and genes involved in the precursors supply, in particular DXS and GGDR, in
Munné-Bosch, 2019). All these genes were expressed in the flavedo of combination with the different isoforms of the gene VTE3.
the three cultivars at harvest and during storage, and changes in their The effect of cold storage on the transcriptional profiling of the genes
transcriptional profile revealed key genes regulating tocopherols syn­ of the different pathways involved in tocopherol synthesis was very
thesis in the flavedo of fruit of the different mandarin genotypes. variable and dependent on each gene and mandarin cultivar (Figs. 4–7).
The precursor HGA, which is common for all tocochromanols, is In general, genes involved in the supply of precursors PPP and HGA,
supplied by the SK pathway, while PPP can either be formed directly with the exception of GGDR and TAT1, were up-regulated by low tem­
from GGPP (produced in the MEP pathway), or from the recycling of peratures, although to a different extent and with variable patterns for

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F. Rey et al. Postharvest Biology and Technology 180 (2021) 111594

each gene. The expression of TAT1 experienced an early repression by accumulation of γ-tocopherol in ‘Nova’ and ‘Nadorcott’.
cold, but increased again at 5–8 weeks of storage (Fig. 6). An induction In summary, our results indicate a positive relationship between total
of HPPD, the other gene involved in HGA synthesis, in response to tocopherol content, particularly α-tocopherol, in the peel of mandarin
different abiotic stresses has been previously described in different plant fruit and the tolerance to develop CI during storage at 2 ◦ C. Higher
species, such as barley, alfalfa and Arabidopsis (Ma et al., 2020). tocopherol content appear to be important for the tolerance of mandarin
Moreover, genes regulating PPP availability, either by the recycling of fruit against cold stress. Expression of 14 genes involved in tocopherol
phytol (VTE5 and VTE6) (Fig. 4) or through the MEP pathway (DXS1, synthesis revealed that the differences in tocopherol content among the
DXS2, GGPPS1 and GGPPS6) (Fig. 6), were stimulated during exposure cultivars are likely related to a higher relative expression of upstream
to 2 ◦ C. Nonetheless, since the induction of these genes occurred in fruit DXS1, DXS2 and GGDR genes, of the MEP pathway, and the gene VTE3b
of the three mandarin genotypes, it is likely that it represents a of the tocopherol-core pathway. While genes of the MEP pathway con­
cold-response not directly related to the varietal susceptibility to CI. The trol the supply of PPP, and thus the influx into the tocopherol-core
only gene differentially affected among cultivars was GGDR, which pathway, the methyltransferase encoded by VTE3b regulates the shift
expression appeared to be related to the genotypic differences in to­ towards γ- and α-tocopherols synthesis. Moreover, the relative balance
copherols content at harvest. GGDR was up-regulated in fruit with low between the expression of upstream genes and VTE4 may also influence
tocopherol content (‘Fortune’) and repressed in the cultivars with higher the accumulation of the different tocopherol sub-forms. Finally, cold
content (‘Nova’ and ‘Nadorcott’) (Fig. 5). Since GGDR regulates the final storage stimulated the expression of most genes of the pathways sup­
step supplying PPP through the MEP pathway, it is tempting to speculate plying tocopherol precursors, and down-regulated those of the
that its up-regulation in the CI-sensitive mandarin may be a CI response. tocopherol-core pathway, but these changes appeared to be cold-related
Genes of the MEP pathway, and in particular DXS and DXR, have been responses rather than CI responses.
shown to be up-regulated in response to different abiotic and biotic
stresses (Xu et al., 2019), probably as part of the mechanisms to provide CRediT authorship contribution statement
precursors for the synthesis of plastidial isoprenoids that may act as
antioxidants under stress conditions. However, in Arabidopsis GGDR Conceptualization, M.J.R. and L.Z.; methodology, F.R, M.J.R. and L.
was not stimulated in response to stress (Collakova and DellaPenna, Z.; experimental work, formal analysis and data curation, F.R; wri­
2003a), similarly to what occurred in ‘Nova’ and ‘Nadorcott’ mandarins. ting—original draft preparation, F.R; writing—review, F.R., M.J.R. and
Exposure to postharvest cold temperatures had a different effect on L.Z.; supervision and funding acquisition, M.J.R. and L.Z. All authors
the expression of the genes of the tocopherol-core pathway, than that have read and agreed to the published version of the manuscript.
observed in genes of the precursor’s pathways. In general, these genes
experienced an early repression that was progressively recovered to­
wards the end of the storage (Fig. 7). This pattern of expression was Declaration of Competing Interest
similar in fruit of the three cultivars, suggesting that they may be cold-
regulated responses rather than associated to the development of CI. The authors declare that they have no known competing financial
Previous studies in Arabidopsis and other model plants have demon­ interests or personal relationships that could have appeared to influence
strated that VTE2, VTE3 and VTE1 genes are essential for the mainte­ the work reported in this paper.
nance of tocopherol levels and the tolerance of plants to different abiotic
stresses, including low temperature (Maeda et al., 2006; Wang et al.,
Acknowledgements
2017; Ma et al., 2020). Moreover, stimulation of these genes is a com­
mon response of plants in response to several stress conditions (Colla­
This work was funded by the research grant RTI2018-095131-B-I00
kova and DellaPenna, 2003a; Ma et al., 2020). In citrus fruit peel,
of the Ministry of Science and Innovation (Spanish Government). F.R. is
however, exposure to low temperatures did not up-regulate the
the recipient of a PhD scholarship (POS_EXT_2016_1_133720) from ANII
expression of these genes in either CI-tolerant nor CI-sensitive cultivars.
(Uruguay). The authors acknowledge the Citrus Germoplasm Bank of
Since tocopherol levels remained nearly constant during cold storage, it
Instituto Valenciano de Investigaciones Agrarias (IVIA, Generalitat
is likely that the up-regulation of upstream genes, as HPPD, VTE6, DXS1
Valenciana) for the provision of fruit. The excellent technical assistance
and GGPPS1, may compensate the down-regulation of most genes of the
of Mª C. Gurrea and I. Carbonell is gratefully acknowledged.
tocopherol-core pathway. The idea that changes in gene expression are
cold-mediated responses was supported by the PCA, in which the first
two components tended to separate individuals according to the pattern Appendix A. Supplementary data
of expression during cold storage, rather than according to the mandarin
cultivar (Fig. 8A and B). A third component differentiated ‘Nadorcott’ Supplementary material related to this article can be found, in the
from the other cultivars, mainly the difference in total and α-tocopherol online version, at doi:https://doi.org/10.1016/j.postharvbio.2021.111
content (Fig. 8C and D). 594.
It is interesting to mention that the changes in the relative abundance
of VTE4 transcripts during storage may account for the differences References
detected in γ-tocopherol among cultivars. In the tolerant ‘Nova’ and
Almeida, J., Quadrana, L., Asís, R., Setta, N., de Godoy, F., Bermúdez, L., Otaiza, S.N.,
‘Nadorcott’ mandarins, but not in the CI-sensitive ‘Fortune’, γ-tocoph­
Corrêa da Silva, J.V., Fernie, A.R., Carrari, F., Rossi, M., 2011. Genetic dissection of
erol increased during cold storage (Fig. 3). Expression of the VTE4 gene, vitamin E biosynthesis in tomato. J. Exp. Bot. 62, 3781–3798. https://doi.org/
responsible of the conversion of γ- to α-tocopherol, was markedly 10.1093/jxb/err055.
Almeida, J., Azevedo, M., da, S., Spicher, L., Glauser, G., vom Dorp, K., Guyer, L., del
repressed in ‘Nova’ and ‘Nadorcott’ mandarins (70 % in both genotypes
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