Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage

International Journal of Molecular Sciences Article Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage Matías Salvo 1, Florencia Rey 2, Ana Arruabarrena 1, Giuliana Gambetta 3, María J. Rodrigo 2, Lorenzo Zacarías 2and Joanna Lado 1,*   Citation: Salvo, M.; Rey, F.; Arruabarrena, A.; Gambetta, G.; Rodrigo, M.J.; Zacarías, L.; Lado, J. Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage. Int. J. Mol. Sci. 2021,22, 804. https://doi.org/10.3390/ijms22020804 Received: 9 December 2020 Accepted: 12 January 2021 Published: 15 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Programa de Investigación en Citricultura, Estación Experimental INIA Salto Grande, Instituto Nacional de Investigación Agropecuaria (INIA), Camino a la Represa s/n, 50000 Salto, Uruguay; [email protected] (M.S.); aarruabarr[email protected] (A.A.) 2 Food Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas (IATA-CSIC), Paterna, 46980 Valencia, Spain; [email protected] (F.R.); [email protected] (M.J.R.); [email protected] (L.Z.) 3Departamento de Producción Vegetal, Facultad de Agronomía, Universidad de la República, Garzón 780, 11900 Montevideo, Uruguay; [email protected] *Correspondence: [email protected]g.uy; Tel.: +598-47335156 Abstract: Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes (CBF1,CBF2, and CBF3) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1–5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus, indicating that CBF1 participates in the natural tolerance to CI. Keywords: CBF; citrus; chilling injury; DREB; gene expression 1. Introduction Citrus is one of the most important fruit crops worldwide, commercialized as fresh fruit or concentrated juice. Export of fresh citrus fruit to certain international markets requires quarantine cold treatments to avoid fruit fly [ 1 ]. However, cold storage (0–1 ◦ C) during long transport could exert negative effects on the fruit of citrus cultivars sensitive to cold. Damage induced by low temperature, known as chilling injury (CI), is usually manifested in the peel, affecting the fruit’s external appearance and commercial quality [ 2 ]. Most characteristic symptoms of postharvest CI in the flavedo (external colored layer of the peel) are manifested as small, depressed areas that progressively become darker and sunken, producing large spots of brown or black color along the fruit surface [2,3]. The incidence of CI in citrus fruit depends on the species, the cultivar, growing conditions, pre-harvest temperatures, as well as fruit maturity at harvest [ 2 , 3 ]. Among the commercial Citrus species, limes, lemons, and grapefruit are considered highly sensitive to CI, more than the fruit of oranges and mandarins. It has been reported that white ‘Marsh’ grapefruit is more sensitive to cold than red-colored ‘Ruby Red’ and ‘Rio Red’ [ 4 ], while Int. J. Mol. Sci. 2021,22, 804. https://doi.org/10.3390/ijms22020804 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 804 2 of 20 ‘Navel’ and ‘Blanca’ oranges are considered more tolerant than the ‘Shamouti’ cultivar [ 2 , 5 ]. In grapefruit, earlier and later harvested fruit are described to be more sensitive to CI than mid-season fruit [ 4 , 6 , 7 ]. By contrast, the opposite pattern of seasonal sensitivity to CI was observed in the cold-sensitive ‘Fortune’ mandarin under Mediterranean conditions [ 8 ], revealing that different pre-harvest factors may modulate fruit tolerance to cold storage. Moreover, we have previously observed that light deprivation in the red ‘Star Ruby‘ grapefruit induced resistance to CI, together with an increased lycopene content and singlet-oxygen antioxidant capacity, indicating the light exposure may directly or indirectly play a role in the tolerance of citrus fruit to CI [9–11]. Because of their subtropical origin, cold stress produces remarkable structural, biochemical, and molecular transformations in the peel of citrus fruit [ 2 ]. Changes in the expression of diverse genes related to a broad array of metabolic functions, such as stress stimuli, transcription factors, hormone biosynthesis, and carbohydrate metabolism, are stimulated or repressed by low temperatures [ 12 – 14 ]. Among transcription factors, C-Repeat Binding Factors (CBFs) have been described as relevant promoters of cold-tolerance associated responses in different cold-sensitive plant species [15], including citrus plants [16–18]. CBFs are transcription factors highly conserved among plants that bind to promoters of genes that respond to low temperatures (COR-cold regulated genes), stimulating their expression and participating in plant acclimation to and survival in low-temperature stress [ 19 – 22 ]. Products of the COR genes were suggested to be relevant in the acquisition of cold tolerance and include transcription factors, protein kinases, late embryogenesis abundant proteins, osmoprotectants, proteins associated with hormone responses, cell wall structure, and lipid metabolisms as well as chloroplastic proteins [ 23 ]. Expression of CBFs genes is stimulated a few hours or even minutes after tissue exposure to low temperatures in different plant organs [ 15 , 24 ]. Moreover, a high transcription of these genes induces cold tolerance in different species [15,25–30], including citrus plants [16,31]. In the model plant Arabidopsis thaliana, different CBFs genes have been reported, and their possible functions explored in relation to cold response and acclimation [ 21 , 32 ]. CBF1 and CBF3 are differentially regulated with respect to CBF2, while CBF4 is not involved in response to low temperatures [ 15 ]. Indeed, the cbf1,2,3triple mutant showed an impaired freezing tolerance after cold exposition, establishing unequivocally that CBF1,2, and 3 genes are important regulators of cold acclimation in Arabidopsis [ 21 ]. Moreover, different ecotypes with contrasting sensitivity to cold exhibited clear differences in the expression of CBF1 and CBF2 genes [ 26 ]. Recent studies indicate that COR genes may also be regulated through CBF-independent pathways [ 23 ]. Evidence suggests that the three CBF proteins are partially redundant regulating COR genes, although some specialization has been inferred by differential expression patterns of these genes [33]. Plants integrate light and temperature signals to respond to changes in the environment. The expression of CBFs genes is also modulated by light in plants. The photoreceptor phytochrome B (phyB) was reported as responsible for the activation of cold-stress signaling in response to light. Light induces CBF1,2, and 3, suggesting that there is a connection between cold and light signaling mediated by phytochromes in Arabidopsis [ 34 ]. Coldinduced CBFs proteins interact with phytochrome-interacting transcription 3 (PIF3) and phyB under cold stress in Arabidopsis, revealing that CBFs stabilize the phyB thermosensor to enhance plant freezing tolerance [ 35 ]. Studies in tomatoes revealed that SlPIF4 directly binds to the promoters of SlCBF genes, and their expression is induced under low temperature via phytochrome A [36]. In Citrus, a possible role of CBFs in the differential cold tolerance of Poncirus trifoliata plants and pummelo (Citrus grandis) has been described since a lower gene expression was found in cold-sensitive pummelo than in Poncirus [ 31 ]. PtCBF expression was induced not only by low temperature but also by abscisic acid [ 17 ], a stress-response phytohormone. Similarly, differences in the cold-induced expression of CBF1 between both species were observed since an earlier, and higher accumulation occurred in leaves of the cold-tolerant Poncirus compared to that of C. paradisi [ 16 ]. Moreover, PtCBF1 putatively regulates Int. J. Mol. Sci. 2021,22, 804 3 of 20 CORc115 expression (a cold-induced group II LEA gene) [ 16 ], which is part of the conserved plant responses to cold [ 23 ]. Therefore, CBFs appear to exert a role in the regulation of cold response in vegetative tissues of Citrus plants; however, information about the potential role of these transcription factors in the responses of fruit to cold during postharvest storage has not been yet addressed. Transcriptional changes in CBF genes during fruit responses to low temperature have been explored in tomatoes, where the expression of SlCBF1 is induced early by low temperatures and is associated with a higher tolerance to low temperatures [ 37 ]. Similarly, CmCBF1 is induced after 6 and 12 h of cold storage, respectively, in the peel and pulp of melon fruit, with higher levels in the cold-tolerant cultivar [ 38 ]. In oil palm fruit ( Elaeis guineensis ), EgCBF3 expression is induced after 2 h of cold treatment with a peak at 24 h [ 24 ]. In peach fruit, the transcription of PpCBF1/5/6 is induced after 12 h of storage at 0 ◦ C and is accompanied by a decrease in CI symptoms, whereas the expression of other CBF genes (PpCBF2/3/4) remains relatively constant [ 28 ]. Ectopic expression of a peach PpCBF1 in apples increased freezing tolerance when compared to the non-transformed control [ 29 ]. Contrastingly, in table grapes, no induction in the expression of VvCBF1 [ 39 ] and VvCBF4/VviDREBA1–1 was observed during storage at 0 ◦ C in the skin nor pulp of the fruit [ 40 , 41 ]. Current studies in Citrus suggest a role for CBFs in plant tolerance to cold under field conditions [ 16 , 31 ], but the involvement of CBFs in the cold tolerance of fruit during postharvest storage has not been explored. Therefore, the objective of this study was to investigate the potential role of CBF genes in the responses of citrus fruit to postharvest storage at low temperature. To unravel that goal, we used fruit of the main Citrus species (lemons, grapefruit, oranges, and mandarins) with contrasting susceptibility to develop CI during cold storage. Since the sensitivity of citrus fruit to CI can be influenced by pre-harvest conditions, such as light incidence during fruit growth [ 2 ], the effect of light deprivation in the expression of CBF genes in the peel of CI-susceptible fruit was also evaluated. 2. Results 2.1. In Silico Study of Citrus CBFs To identify all members of the CBFs family in Citrus, we first carried out a BLASTP search of the Citrus sinensis (sweet orange) genome database at Phytozome 12 (JGI, Sweet Orange Genome Project Citrus sinensis v1.1 https://phytozome.jgi.doe.gov/) with the Arabidopsis CBFs (AtCBF1; AT4G25490.1; AtCBF2, AT4G25470.1; AtCBF3, AT4G25480.1). AtCBF4 was excluded from this analysis since it is involved in drought stress responses rather than cold [42]. The analysis revealed the presence of three genes encoding CBFs in Citrus:CBF1 (orange1.1g028094m), CBF2 (orange1.1g026103m), and CBF3 (orange1.1g029015m). The length of predicted proteins was 214, 243, and 201 amino acids for CBF1,2, and 3, respectively, and the C-terminal of all three proteins showed an acid isoelectric point as reported for dicot CBFs [ 40 ]. The search for functional and structural domains in the Citrus CBFs displayed most of the characteristic CBF features, although not all of them were fully conserved in all members (Figure 1). CBF1 and CBF2 showed the N-terminal PEST [peptide sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] domain (https://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind) , which is present in other members of the DREB (dehydration-responsive element binding) subfamily and has been associated with rapid protein turnover by targeting proteolytic degradation [ 40 , 43 ]. The CBFconserved domains PKKRAGR (DREBA1 signature sequence PKKP/RAGRxKFxETRHP) and DSAWR (DREBA1 signature sequence DS(A/V/S)WRL) flanking the AP2 (Apetala2) domain were present in the three Citrus CBFs, but the full consensus sequences of both domains PKKRAGR and DSAWR were only conserved in Citrus CBF1 and 3, respectively (Figure 1 and Figure S1). The AP2 typical domain [ 44 ] showed a high degree of sequence identity with other plant CBFs (Figure 1and Figure S1). The AP2 characteristic WLG and RAHD motifs, and valine (position14) and glutamic acid (position 19) were conserved in all citrus CBFs, but the YRG motif was only fully conserved in citrus CBF1 (Figure S1). The AP2 down- Int. J. Mol. Sci. 2021,22, 804 4 of 20 stream A(A/V)xxA(A/V)xxF sequence conserved in all DREBA1 homologs [ 45 ] was also identified in the three Citrus CBF, and the C-terminal LWSY motif [ 46 ] was only conserved in the Citrus CBF1 (Figure 1and Figure S1). The hydrophobic cluster analysis (HCA) of the C-terminus was performed (http://bioserv.rpbs.univ-paris-diderot.fr/services/HCA/) and showed that all Citrus CBFs contained five hydrophobic clusters, which has been described as important for trans-activation of target genes [47]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 20 Citrus CBF1 and 3, respectively (Figure 1 and FigureS1). The AP2 typical domain [44] showed a high degree of sequence identity with other plant CBFs (Figure 1 and Figure S1). The AP2 characteristic WLG and RAHD motifs, and valine (position14) and glutamic acid (position 19) were conserved in all citrus CBFs, but the YRG motif was only fully conserved in citrus CBF1 (Figure S1). The AP2 downstream A(A/V)xxA(A/V)xxF sequence conserved in all DREBA1 homologs [45] was also identified in the three Citrus CBF, and the C-terminal LWSY motif [46] was only conserved in the Citrus CBF1 (Figure 1 and Figure S1). The hydrophobic cluster analysis (HCA) of the C-terminus was performed (http://bioserv.rpbs.univ-paris-diderot.fr/services/HCA/) and showed that all Citrus CBFs contained five hydrophobic clusters, which has been described as important for trans-activation of target genes [47]. Figure 1. Schematic representation of C-repeat binding factors (CBFs) from Citrus sinensis (CBF1, orange1.1g028094m; CBF2 orange1.1g026103m; CBF3, orange1.1g029015m) showing the main characteristics domains. The PEST motif (grey), the PKKPAGR (dehydration-responsive element binding (DREB) A 1 signature sequence PKKP/RAGRxKFxETRHP) motif (pink), the AP2 domain (yellow), the DSAWRL (DREBA1 signature sequence DS(A/V/S)WRL) motif (blue), and the A(A/V)xxA(A/V)xxF motif (green) are present in the citrus CBFs. The C-terminus hydrophobic clusters (HC2-HC6) (gray) are indicated. The C-terminal LWSY motif was only identified in citrus CBF1. The comparison of the full protein sequences of Citrus CBFs revealed that CBF2 and 3 were the most closely related sequences (72.8% identity), and both were least similar to CBF1 (about 60% identity) (Table S1). The relationship between the Citrus CBFs with other plant CBFs proteins, including three members from Arabidopsis, tomatoes, and table grapes, was analyzed by sequence comparison and by the generation of a phylogenetic tree (Figure 2). The Citrus CBF1 grouped in a cluster with table grape VvDREBA1–1, and this cluster was grouped with tomatoes and Arabidopsis CBFs (Figure 2). Interestingly, Citrus CBF2 and 3 were located together in a separate branch and more distantly related to other CBFs (Figure 2). The comparison of full sequences of Citrus CBFs with other plant homologs showed that Citrus CBF1 displayed a slightly higher percentage of identity with Arabidopsis, tomatoes, and table grape members, ranging from 49% to 67%, in comparison to CBF2 and CBF3 (43% to 62% of identity) (Table S2). Figure 1. Schematic representation of C-repeat binding factors (CBFs) from Citrus sinensis (CBF1, orange1.1g028094m; CBF2 orange1.1g026103m; CBF3, orange1.1g029015m) showing the main characteristics domains. The PEST motif (grey), the PKKPAGR (dehydration-responsive element binding (DREB) A 1 signature sequence PKKP/RAGRxKFxETRHP) motif (pink), the AP2 domain (yellow), the DSAWRL (DREBA1 signature sequence DS(A/V/S)WRL) motif (blue), and the A(A/V)xxA(A/V)xxF motif (green) are present in the citrus CBFs. The C-terminus hydrophobic clusters (HC2-HC6) (gray) are indicated. The C-terminal LWSY motif was only identified in citrus CBF1. The comparison of the full protein sequences of Citrus CBFs revealed that CBF2 and 3were the most closely related sequences (72.8% identity), and both were least similar to CBF1 (about 60% identity) (Table S1). The relationship between the Citrus CBFs with other plant CBFs proteins, including three members from Arabidopsis, tomatoes, and table grapes, was analyzed by sequence comparison and by the generation of a phylogenetic tree (Figure 2). The Citrus CBF1 grouped in a cluster with table grape VvDREBA1–1, and this cluster was grouped with tomatoes and Arabidopsis CBFs (Figure 2). Interestingly, Citrus CBF2 and 3 were located together in a separate branch and more distantly related to other CBFs (Figure 2). The comparison of full sequences of Citrus CBFs with other plant homologs showed that Citrus CBF1 displayed a slightly higher percentage of identity with Arabidopsis, tomatoes, and table grape members, ranging from 49% to 67%, in comparison to CBF2 and CBF3 (43% to 62% of identity) (Table S2). Int. J. Mol. Sci. 2021,22, 804 5 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 20 Figure 2. Phylogenetic tree of Citrus CBFs and other plant CBFs. The phylogenetic tree was generated based on the alignment of deduced amino acid sequences of Citrus sinensis CBF1, 2, and 3 proteins and Arabidopsis, tomatoes, and table grapes CBFs. The tree was constructed based on the Neighbor-Joining method [48]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [49]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The sequences used to generate the phylogenetic tree and their accession numbers are as follows: Citrus sinensis CBF1 (orange1.1g028094m), CBF2 (orange1.1g026103m), and CBF3 (orange1.1g029015m); Arabidopsis thaliana AtCBF1 (AT4G25490.1), AtCBF2 (AT4G25470.1) and AtCBF3 (AT4G25480.1); Solanum lycopersicum SlCBF1 (Q8S9N5), SlCBF2 (XP_004234350.1) and SlCBF3 (AAS77819.1); Vitis vinifera VviDREBA1–6 (MF445008), VviDREBA1–7 (MF445009) and VviDREBA1–1 (MF445007). Evolutionary analysis was conducted in MEGA7 [50]. 2.2. CI Symptoms and Expression of CBFs Genes in Cold-Tolerant and Cold-Sensitive Citrus Fruits during Cold Storage CI incidence was evaluated in the fruit of two cultivars of the most important Citrus species: lemons, grapefruit, oranges, and mandarins. Both cultivars showed contrasting sensitivity to CI during storage at 1 °C for two months (Figures 3–6). In lemons, the fruit of Lisbon were more resistant to CI than those of Meyer, since after 58 d of storage CI, the index in Lisbon was about 0.28, whereas in Meyer, it was 2.5 (Figure 3A). Initial CI symptoms appeared in the peel of Meyer after 14 d of cold exposure, showing brown depressed areas that progressively increased in extension and developed large clustered brown areas. By contrast, the fruit of the Lisbon cultivar only developed small scattered pits on the fruit surface (Figure 3A). Grapefruit cultivars also showed differences in CI incidence, with lower levels in Star Ruby than in Marsh fruit: 1.57 and 2.81 after 58 d of storage, respectively. Marsh symptoms appeared as early as 14 d after storage (Figure 4A). Sweet orange fruits also showed contrasting sensitivity to CI, Navelina being more tolerant than Salustiana (CI index of 0.17 and 1.96, respectively, at the end of the storage period). CI symptoms in orange fruit developed as discrete sunken areas that progressively became bronze, covering a wide surface of the fruit (Figure 5A). A comparison of CI between the fruit of Fortune and Nadorcott mandarins also revealed marked differences in Figure 2. Phylogenetic tree of Citrus CBFs and other plant CBFs. The phylogenetic tree was generated based on the alignment of deduced amino acid sequences of Citrus sinensis CBF1,2, and 3proteins and Arabidopsis, tomatoes, and table grapes CBFs. The tree was constructed based on the Neighbor-Joining method [ 48 ]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [ 49 ]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The sequences used to generate the phylogenetic tree and their accession numbers are as follows: Citrus sinensis CBF1 (orange1.1g028094m), CBF2 (orange1.1g026103m), and CBF3 (orange1.1g029015m); Arabidopsis thaliana AtCBF1 (AT4G25490.1), AtCBF2 (AT4G25470.1) and AtCBF3 (AT4G25480.1); Solanum lycopersicum SlCBF1 (Q8S9N5), SlCBF2 (XP_004234350.1) and SlCBF3 (AAS77819.1); Vitis vinifera VviDREBA1–6 (MF445008), VviDREBA1–7 (MF445009) and VviDREBA1–1 (MF445007). Evolutionary analysis was conducted in MEGA7 [50]. 2.2. CI Symptoms and Expression of CBFs Genes in Cold-Tolerant and Cold-Sensitive Citrus Fruits during Cold Storage CI incidence was evaluated in the fruit of two cultivars of the most important Citrus species: lemons, grapefruit, oranges, and mandarins. Both cultivars showed contrasting sensitivity to CI during storage at 1 ◦ C for two months (Figures 3–6). In lemons, the fruit of Lisbon were more resistant to CI than those of Meyer, since after 58 d of storage CI, the index in Lisbon was about 0.28, whereas in Meyer, it was 2.5 (Figure 3A). Initial CI symptoms appeared in the peel of Meyer after 14 d of cold exposure, showing brown depressed areas that progressively increased in extension and developed large clustered brown areas. By contrast, the fruit of the Lisbon cultivar only developed small scattered pits on the fruit surface (Figure 3A). Grapefruit cultivars also showed differences in CI incidence, with lower levels in Star Ruby than in Marsh fruit: 1.57 and 2.81 after 58 d of storage, respectively. Marsh symptoms appeared as early as 14 d after storage ( Figure 4A ). Sweet orange fruits also showed contrasting sensitivity to CI, Navelina being more tolerant than Salustiana (CI index of 0.17 and 1.96, respectively, at the end of the storage period). CI symptoms in orange fruit developed as discrete sunken areas that progressively became bronze, covering a wide surface of the fruit (Figure 5A). A comparison of CI between the fruit of Fortune and Nadorcott mandarins also revealed marked differences in susceptibility to CI. Fortune fruit were very susceptible to CI (2.90 after 58 d), while Nadorcott fruit were Int. J. Mol. Sci. 2021,22, 804 6 of 20 highly tolerant during the whole storage period (0.26 after 58 d). The onset of chilling symptoms in Fortune was detected after 21 d of storage and manifested as the typical pitting symptoms speared over the fruit surface, whereas Nadorcott mandarins were almost devoid of damage (Figure 6A). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 20 susceptibility to CI. Fortune fruit were very susceptible to CI (2.90 after 58 d), while Nadorcott fruit were highly tolerant during the whole storage period (0.26 after 58 d). The onset of chilling symptoms in Fortune was detected after 21 d of storage and manifested as the typical pitting symptoms speared over the fruit surface, whereas Nadorcott mandarins were almost devoid of damage (Figure 6A). Figure 3. (A) Chilling injury (CI) index in Lisbon and Meyer lemons at harvest and during cold storage at 1 °C and (B) relative expression of CBF1, CBF2, and CBF3 in Lisbon (black bars) and Meyer (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p < 0.05). Figure 3. ( A ) Chilling injury (CI) index in Lisbon and Meyer lemons at harvest and during cold storage at 1 ◦ C and ( B ) relative expression of CBF1,CBF2, and CBF3 in Lisbon (black bars) and Meyer (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p< 0.05). Int. J. Mol. Sci. 2021,22, 804 7 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 20 Figure 4. (A) CI index in Star Ruby and Marsh grapefruit at harvest and during cold storage at 1 °C and (B) relative expression of CBF1, CBF2, and CBF3 in Star Ruby (black bars) and Marsh (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p < 0.05). Figure 4. ( A ) CI index in Star Ruby and Marsh grapefruit at harvest and during cold storage at 1 ◦ C and ( B ) relative expression of CBF1,CBF2, and CBF3 in Star Ruby (black bars) and Marsh (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p< 0.05). Int. J. Mol. Sci. 2021,22, 804 8 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 20 Figure 5. (A) CI index in Navelina and Salustiana oranges at harvest and during cold storage at 1 °C and (B) relative expression of CBF1, CBF2, and CBF3 in Navelina (black bars) and Salustiana (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p < 0.05). Figure 5. ( A ) CI index in Navelina and Salustiana oranges at harvest and during cold storage at 1 ◦ C and ( B ) relative expression of CBF1,CBF2, and CBF3 in Navelina (black bars) and Salustiana (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p< 0.05). Int. J. Mol. Sci. 2021,22, 804 9 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 20 Figure 6. (A) CI index in Nadorcott and Fortune mandarins at harvest and during cold at 1 °C and (B) relative expression of CBF1, CBF2, and CBF3 in Nadorcott (black bars) and Fortune (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p < 0.05). The expression of the three CBF genes (CBF1, CBF2, and CBF3) in the flavedo of the fruit of the eight Citrus cultivars was evaluated during 58 d of cold storage. Lisbon lemon showed an early induction of CBF1 after cold exposure (1 and 5 d), decreasing afterward (except for 35 d), whereas, in the Meyer cultivar, its expression decreased. The expression of CBF2 and CBF3 decreased in Lisbon, especially at 1 d, 28 d, and 58 d, while Meyer showed a transient increase in CBF3 after 5 d of cold storage (Figure 3B). In Star Ruby grapefruit, expression of the three CBF genes increased after 1 and 5 d, CBF1 being much higher than that of CBF2 and CBF3 (10, 1.5, and 2-times higher than the initial, respectively). In contrast, the expression in Marsh grapefruit remained almost unchanged during cold storage for CBF1 but decreased in CBF2 and CBF3 (Figure 4B). In Navelina orange, Figure 6. ( A ) CI index in Nadorcott and Fortune mandarins at harvest and during cold at 1 ◦ C and ( B ) relative expression of CBF1,CBF2, and CBF3 in Nadorcott (black bars) and Fortune (grey bars) during cold storage (means ± S.E.). Pictures show the external appearance of fruit at 58 d of cold storage. For each cultivar, asterisks indicate significant differences in the expression of a CBF gene between each time-point and the harvest time (which were set to 1), by a Student’s t-test (p< 0.05). The expression of the three CBF genes (CBF1,CBF2, and CBF3) in the flavedo of the fruit of the eight Citrus cultivars was evaluated during 58 d of cold storage. Lisbon lemon showed an early induction of CBF1 after cold exposure (1 and 5 d), decreasing afterward (except for 35 d), whereas, in the Meyer cultivar, its expression decreased. The expression of CBF2 and CBF3 decreased in Lisbon, especially at 1 d, 28 d, and 58 d, while Meyer showed a transient increase in CBF3 after 5 d of cold storage (Figure 3B). In Star Ruby grapefruit, expression of the three CBF genes increased after 1 and 5 d, CBF1 being much higher than that of CBF2 and CBF3 (10, 1.5, and 2-times higher than the initial, respectively). In contrast, the expression in Marsh grapefruit remained almost unchanged Int. J. Mol. Sci. 2021,22, 804 16 of 20 postharvest performance in covered and non-covered fruits was conducted in independent experiments in different seasons (2018 and 2019), representing the average of both seasons. 4.2. Fruit Color and Chilling Injury Evaluation At harvest and during storage, peel color of whole fruit was measured using a Minolta CR-400 colorimeter (Minolta, USA) on three areas of the equatorial plane of the fruit and expressed as the ICC (citrus color index), calculated with Formula (1). A lower ICC value (more negative) represents green fruit, near-zero values correspond to yellow fruit at the color break, and orangeto red-colored fruit reflects positive values. ICC = (1000 ×a)/(L ×b), (1) Fruit were inspected for CI symptoms (intensity and extension of the damage) after 1, 5, 14, 28, 35, and 58 d in cold storage. The severity of the symptoms was assessed visually using the following scale: 0 = no pitting; 1 = pitting covering <25% of the fruit surface; 2 = pitting covering between 25 and 50% of the surface; 3 = 50–100%. CI index was calculated using the Formula (2) described in [10]: CI index =∑[(CI level)×(Number of fruits at the CI level)] Total number of fruits evaluated (2) The experimental design was completely randomized, and results correspond to the mean ±S.E. of four replicates of 20 fruit each. 4.3. RNA Extraction and Quantitative Real-Time PCR Analysis Total RNA was isolated from plant material following the protocol described in [ 64 ] with modifications. Two tubes of 2 mL each with 0.2 g of flavedo tissue were processed by sample. 770 µ L of extraction buffer (200 mM Tris-HCl, pH 8.0, 400 mM NaCl, 50 mM Na2EDTA, 2% (w/v) Sarkosyl, 1% (w/v) poly(vinylpyrrolidone), and 1% (v/v) βmercaptoethanol), and 380 µ L of phenol was added to each tube. Tubes were vortexed and incubated at 65 ◦ C for 15 min. Three hundred and eighty microliters of chloroform:isoamilic alcohol (24:1) were added to each tube and centrifuged at 4000 × g, 10 min at room temperature. The aqueous phase was transferred to a new 2 mL tube and reextracted with 380 µ L of phenol and 380 µ L of chloroform:isoamilic alcohol (24:1). Tubes were centrifuged at 4000 × gfor 10 min, the aqueous phase was transferred to a new 1.5 mL tube, and RNA was precipitated with 1.5 vol of ethanol. After precipitation, tubes were centrifuged at 20,000×g for 30 min at 4 ◦ C. The pellet was washed with 500 µ L of 70% ethanol and resuspended with 300 µ L of ultrapure RNAse-free water. Replicate tubes from the same sample were mixed, 1/3 vol of LiCl 12 M was added, and tubes were incubated on ice at 4 ◦ C overnight. Tubes were centrifuged at 20,000 × gfor 30 min at 4 ◦ C, and the pellet was washed with 800 µ L of 70% ethanol. The pellet was dried at room temperature and resuspended in 50 µ L of ultrapure RNAse-free water. RNA was quantified, with a recording absorbance at 260 nm, and quality was verified by sample absorbance at 260/280 nm and 260/230 nm. The integrity of RNA was evaluated by 1% agarose gel electrophoresis. For each sample, 10 µ g of RNA was treated with a DNAse Turbo DNA-free TM kit (ThermoFisher Scientific, Lithuania) according to the manufacturer’s instructions. After DNAse treatment, cDNA synthesis was performed with 1 µ g of treated RNA and using a RevertAid Reverse transcriptase kit (Thermo Scientific, Lithuania) according to the manufacturer’s specifications. Quantitative RT-PCR reactions were performed using an Applied Biosystems StepOne TM Plus Real-Time PCR System (Applied Biosystems, San Francisco, CA, USA). Each reaction consisted of 2 µ L of a dilution 1:4 of cDNA, 1 µ L of primer mix (10 µ M each), and 10 µ L of SensiFAST ™ SYBR ® —HiRox kit (Bioline, UK). Primers used for amplification of Actin,CBF1, CBF2, and CBF3 are listed in Table S1. The cycling condition for all genes analyzed consisted Int. J. Mol. Sci. 2021,22, 804 17 of 20 of 10 min at 95 ◦ C for pre-incubation, 40 cycles of 15 s at 95 ◦ C, 15 s at 59 ◦ C, and 15 s at 72 ◦C. Fluorescence intensity data were acquired during the extension step. The specificity of the PCR reaction was confirmed by the presence of a single peak in the dissociation curve performed after the amplification steps. Relative expression was determined using the Pfaffl method [ 65 ], where gene expression was normalized using the expression levels of Actin, a constitutive gene, in the assay conditions [ 66 ]. For all genes and cultivars analyzed, the reference sample was harvest condition. All gene expression data were represented as the mean of three replicates ± SE. Gene expression was analyzed using the Student’s t-test, being the difference between harvest time (0 d, which were set at 1) and each sampling time (1, 5, 28, 35, or 58 d) considered significant when p< 0.05 in a two-tailed analysis. Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/2/804/s1, Table S1. qRT-PCR primer sequences; Table S2. Percent identity matrix of deduced amino acid sequences of CBFs from sweet orange, Arabidopsis, table grapes, and tomatoes; Table S3. Fruit color (ICC values) at harvest time and during cold storage of Meyer, Marsh, Salustiana, and Fortune non-covered and covered fruit. Figure S1. Alignment of Citrus sinensis CBF proteins and other plant CBFs. Author Contributions: Conceptualization, M.J.R., L.Z., J.L.; methodology and experimental, M.S., F.R., A.A., G.G.; formal analysis, M.S., F.R., L.Z., M.J.R., J.L.; writing—original draft preparation, M.S., J.L.; writing— review and editing, G.G., A.A., F.R., M.J.R., L.Z., J.L.; supervision, A.A., J.L., M.J.R., L.Z.; funding acquisition, J.L., M.J.R., L.Z. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a research grant from the National Agency of Research and Innovation (ANII-Uruguay) by the code FCE_3_2016_1_126714 and by a research grant RTI2018– 095131-B-I00 of the Ministry of Science and Innovation (Spanish Government). F. Rey is the recipient of a predoctoral scholarship (POS_EXT_2016_1_133720) from ANII (Uruguay). Data Availability Statement: The data presented in this study are available in the article or supplementary material. Acknowledgments: We gratefully acknowledge the support from the INIA team Pedro Pintos, Eleana Luque, and Ana Inés Moltini. F.R., M.J.R., and L.Z. are members of the CaRed Excellence Network (BIO2017–90877-REDT). Conflicts of Interest: The authors declare that they have no conflict of interest. Abbreviations ABA Abscisic acid AP2 Apetala2 CI Chilling injury CBFs C-repeat binding factors COR genes Cold regulated genes ICC Citrus color index phyB Phytochrome B PIF3 Phytochrome-interacting transcription 3 DREB Dehydration-responsive element binding HCA Hydrophobic cluster analysis References 1. Biolatto, A.; Vazquez, D.E.; Sancho, A.M.; Carduza, F.J.; Pensel, N.A. Effect of commercial conditioning and cold quarantine storage treatments on fruit quality of “Rouge La Toma” grapefruit (Citrus paradisi Macf.). Postharvest Biol. Technol. 2005 ,35, 167–176. [CrossRef] 2. Lado, J.; Cronje, P.J.; Rodrigo, M.J.; Zacarías, L. Citrus. In Postharvest Physiological Disorders in Fruits and Vegetables; de Freitas, S.T., Sunil, P., Eds.; CRC Press: Boca Raton, FL, USA; Taylor & Francis: Abingdon, UK, 2019; pp. 377–398. ISBN 9781315267470. 3. Lafuente, M.T.; Zacarías, L. Postharvest physiological disorders in citrus fruit. Stewart Postharvest Rev. 2006,2, 1–9. [CrossRef] Int. J. Mol. Sci. 2021,22, 804 18 of 20 4. Dou, H. The influence of harvesting time and geographical location on susceptibility to physiological peel disorders associated with four Florida grapefruit cultivars. J. Hortic. Sci. Biotechnol. 2005,80, 399–402. [CrossRef] 5. Chalutz, E.; Waks, J.; Schiffmann-Nadel, M. A comparison of the response of different citrus fruit cultivars to storage temperature. Sci. Hortic. (Amsterdam) 1985,25, 271–277. [CrossRef] 6. Purvis, A.C. Relationship between mid-season resistance to chilling injury and reducing sugar level in grapefruit peel. HortScience 1979,14, 227–229. 7. Schirra, M.; Agabbio, M.; D’Hallewin, G. Chilling responses of grapefruit as affected by cultivar and harvest date. Adv. Hortic. Sci. 1998,12, 118–122. 8. Lafuente, M.T.; Martínez-Téllez, M.A.; Zacarías, L. Abscisic Acid in the Response of ‘Fortune’ Mandarins to Chilling. Effect of Maturity and High-Temperature Conditioning. J. Sci. Food Agric. 1997,73, 494–502. [CrossRef] 9. Lado, J.; Cronje, P.; Alquézar, B.; Page, A.; Manzi, M.; Gómez-Cadenas, A.; Stead, A.D.; Zacarías, L.; Rodrigo, M.J. Fruit shading enhances peel color, carotenes accumulation and chromoplast differentiation in red grapefruit. Physiol. Plant. 2015 ,154, 469–484. [CrossRef] 10. Lado, J.; Rodrigo, M.J.; Cronje, P.; Zacarías, L. Involvement of lycopene in the induction of tolerance to chilling injury in grapefruit. Postharvest Biol. Technol. 2015,100, 176–186. [CrossRef] 11. Lado, J.; Rodrigo, M.J.; López-Climent, M.; Gómez-Cadenas, A.; Zacarías, L. Implication of the antioxidant system in chilling injury tolerance in the red peel of grapefruit. Postharvest Biol. Technol. 2016,111, 214–223. [CrossRef] 12. Sapitnitskaya, M.; Maul, P.; McCollum, G.T.; Guy, C.L.; Weiss, B.; Samach, A.; Porat, R. Postharvest heat and conditioning treatments activate different molecular responses and reduce chilling injuries in grapefruit. J. Exp. Bot. 2006 ,57, 2943–2953. [CrossRef] [PubMed] 13. Zhu, A.; Li, W.; Ye, J.; Sun, X.; Ding, Y.; Cheng, Y.; Deng, X. Microarray Expression Profiling of Postharvest Ponkan Mandarin (Citrus reticulata) Fruit under Cold Storage Reveals Regulatory Gene Candidates and Implications on Soluble Sugars Metabolism. J. Integr. Plant. Biol. 2011,53, 358–374. [CrossRef] [PubMed] 14. Maul, P.; McCollum, G.T.; Popp, M.; Guy, C.L.; Porat, R. Transcriptome profiling of grapefruit flavedo following exposure to low temperature and conditioning treatments uncovers principal molecular components involved in chilling tolerance and susceptibility. Plant Cell Environ. 2008,31, 752–768. [CrossRef] [PubMed] 15. Zhou, M.Q.; Shen, C.; Wu, L.H.; Tang, K.X.; Lin, J. CBF-dependent signaling pathway: A key responder to low temperature stress in plants. Crit. Rev. Biotechnol. 2011,31, 186–192. [CrossRef] [PubMed] 16. Champ, K.I.; Febres, V.J.; Moore, G.A. The role of CBF transcriptional activators in two Citrus species (Poncirus and Citrus) with contrasting levels of freezing tolerance. Physiol. Plant 2007,129, 529–541. [CrossRef] 17. He, L.G.; Wang, H.L.; Liu, D.C.; Zhao, Y.J.; Xu, M.; Zhu, M.; Wei, G.Q.; Sun, Z.H. Isolation and expression of a cold-responsive gene PtCBF in Poncirus trifoliata and isolation of citrus CBF promoters. Biol. Plant. 2012,56, 484–492. [CrossRef] 18. He, L.; Jiang, Y.; Wang, H.; Xu, M.; Sun, Z. Expression and regulation of a cold-responsive gene, CsCBF in Citrus sinensis (L.) Osbeck under low temperature, high salinity and abscisic acid. Acta Hortic. 2016,1135, 33–46. [CrossRef] 19. Chinnusamy, V.; Zhu, J.K.; Sunkar, R. Gene regulation during cold stress acclimation in plants. In Pllant Stress Tolerance. Methods in Molecular Biology; Sunkar, R., Ed.; Humana Press: Totowa, NJ, USA, 2010; Volume 639, pp. 39–55; ISBN 978-1-60761-701-3. 20. Miura, K.; Furumoto, T. Cold signaling and cold response in plants. Int. J. Mol. Sci. 2013,14, 5312–5337. [CrossRef] 21. Jia, Y.; Ding, Y.; Shi, Y.; Zhang, X.; Gong, Z.; Yang, S. The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol. 2016,212, 345–353. [CrossRef] 22. Zhang, X.; Fowler, S.G.; Cheng, H.; Lou, Y.; Rhee, S.Y.; Stockinger, E.J.; Thomashow, M.F. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant. J. 2004 , 39, 905–919. [CrossRef] 23. Liu, Y.; Dang, P.; Liu, L.; He, C. Cold acclimation by the CBF-COR pathway in a changing climate: Lessons from Arabidopsis thaliana. Plant. Cell Rep. 2019,38, 511–519. [CrossRef] [PubMed] 24. Ebrahimi, M.; Abdullah, S.N.A.; Aziz, M.A.; Namasivayam, P. A novel CBF that regulates abiotic stress response and the ripening process in oil palm (Elaeis guineensis) fruits. Tree Genet. Genomes 2015,11. [CrossRef] 25. Qin, F.; Sakuma, Y.; Li, J.; Liu, Q.; Li, Y.-Q.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant. Cell Physiol. 2004 ,45, 1042–1052. [CrossRef] [PubMed] 26. Mizoi, J.; Shinozaki, K.; Yamaguchi-Shinozaki, K. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim. Biophys. Acta 2012,1819, 86–96. [CrossRef] [PubMed] 27. Hu, Y.; Jiang, L.; Wang, F.; Yu, D. Jasmonate Regulates the INDUCER OF CBF EXPRESSION-C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis. Plant. Cell 2013,25, 2907–2924. [CrossRef] 28. Liang, L.; Zhang, B.; Yin, X.-R.; Xu, C.-J.; Sun, C.-D.; Chen, K.-S. Differential Expression of the CBF Gene Family During Postharvest Cold Storage and Subsequent Shelf-Life of Peach Fruit. Plant Mol. Biol. Rep. 2013,31, 1358–1367. [CrossRef] 29. Wisniewski, M.; Norelli, J.; Artlip, T. Overexpression of a peach CBF gene in apple: A model for understanding the integration of growth, dormancy, and cold hardiness in woody plants. Front. Plant Sci. 2015,6, 1–13. [CrossRef] 30. Ahmad, M.; Li, J.; Yang, Q.; Jamil, W.; Teng, Y.; Bai, S. Phylogenetic, Molecular, and Functional Characterization of PpyCBF Proteins in Asian Pears (Pyrus pyrifolia). Int. J. Mol. Sci. 2019,20, 2074. [CrossRef] Int. J. Mol. Sci. 2021,22, 804 19 of 20 31. ¸Sahin-Çevik, M.; Moore, G.A. Two AP2 domain containing genes isolated from the cold-hardy Citrus relative Poncirus trifoliata are induced in response to cold. Funct. Plant Biol. 2006,33, 863. [CrossRef] 32. Liu, J.; Shi, Y.; Yang, S. Insights into the regulation of C-repeat binding factors in plant cold signaling. J. Integr. Plant Biol. 2018 ,60, 780–795. [CrossRef] 33. Shi, Y.; Huang, J.; Sun, T.; Wang, X.; Zhu, C.; Ai, Y.; Gu, H. The precise regulation of different COR genes by individual CBF transcription factors in Arabidopsis thaliana. J. Integr. Plant Biol. 2017,59, 118–133. [CrossRef] [PubMed] 34. Kim, H.-J.; Kim, Y.-K.; Park, J.-Y.; Kim, J. Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. Plant J. 2002 ,29, 693–704. [CrossRef] [PubMed] 35. Jiang, B.; Shi, Y.; Peng, Y.; Jia, Y.; Yan, Y.; Dong, X.; Li, H.; Dong, J.; Li, J.; Gong, Z.; et al. Cold-induced CBF–PIF3 interaction enhances freezing tolerance by stabilizing the phyB thermosensor in Arabidopsis. Mol. Plant 2020 ,13, 894–906. [CrossRef] [PubMed] 36. Wang, F.; Chen, X.; Dong, S.; Jiang, X.; Wang, L.; Yu, J.; Zhou, Y. Crosstalk of PIF4 and DELLA modulates CBF transcript and hormone homeostasis in cold response in tomato. Plant Biotechnol. J. 2019,2, 1–15. [CrossRef] [PubMed] 37. Zhao, D.; Shen, L.; Fan, B.; Yu, M.; Zheng, Y.; Lv, S.; Sheng, J. Ethylene and cold participate in the regulation of LeCBF1 gene expression in postharvest tomato fruits. FEBS Lett. 2009,583, 3329–3334. [CrossRef] [PubMed] 38. Zhang, T.; Zhang, Q.; Pan, Y.; Che, F.; Wang, Q.; Meng, X.; Rao, J. Changes of polyamines and CBFs expressions of two Hami melon (Cucumis melo L.) cultivars during low temperature storage. Sci. Hortic. (Amsterdam) 2017,224, 8–16. [CrossRef] 39. Xiao, H.; Siddiqua, M.; Braybrook, S.; Nassuth, A. Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid. Plant Cell Environ. 2006,29, 1410–1421. [CrossRef] 40. Vazquez-Hernandez, M.; Romero, I.; Escribano, M.I.; Merodio, C.; Sanchez-Ballesta, M.T. Deciphering the Role of CBF/DREB Transcription Factors and Dehydrins in Maintaining the Quality of Table Grapes cv. Autumn Royal Treated with High CO 2 Levels and Stored at 0 ◦C. Front. Plant Sci. 2017,8, 1591. [CrossRef] 41. Fernandez-Caballero, C.; Rosales, R.; Romero, I.; Escribano, M.I.; Merodio, C.; Sanchez-Ballesta, M.T. Unraveling the roles of CBF1, CBF4 and dehydrin 1 genes in the response of table grapes to high CO2 levels and low temperature. J. Plant Physiol. 2012 , 169, 744–748. [CrossRef] 42. Haake, V.; Cook, D.; Riechmann, L.; Pineda, O.; Thomashow, M.F.; Zhang, J.Z. Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis. Plant Physiol. 2002,130, 639–648. [CrossRef] 43. Gregorio, J.; Hernández-bernal, A.F.; Cordoba, E.; León, P. Characterization of evolutionarily conserved motifs involved in activity and regulation of the ABA-INSENSITIVE (ABI) 4 transcription factor. Mol. Plant 2014 ,7, 422–436. [CrossRef] [PubMed] 44. Akhtar, M.; Jaiswal, A.; Taj, G.; Jaiswal, J.P.; Qureshi, M.I.; Singh, N.K. DREB1/CBF transcription factors: Their structure, function and role in abiotic stress tolerance in plants. J. Genet. 2012,91, 385–395. [CrossRef] [PubMed] 45. Xiong, Y.; Fei, S. Functional and phylogenetic analysis of a DREB/CBF-like gene in perennial ryegrass (Lolium perenne L.). Planta 2006,224, 878–888. [CrossRef] [PubMed] 46. Dubouzet, J.; Sakuma, Y.; Ito, Y.; Kasuga, M.; Dubouzet, E.; Miura, S.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. OsDREB genes in rice Oryza sativa L., encode transcription activators that function in drought-, high-saltand cold-responsive gene expression. Plant J. 2003,33, 751–763. [CrossRef] [PubMed] 47. Wang, Z.; Triezenberg, S.J.; Thomashow, M.F.; Stockinger, E.J. Multiple hydrophobic motifs in Arabidopsis CBF1 COOH-terminus provide functional redundancy in trans-activation. Plant Mol. Biol. 2005, 543–559. [CrossRef] 48. Saitou, N.; Nei, M. The Neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987 ,4, 406–425. 49. Felsenstein, J. Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution (N. Y). 1985,39, 783. [CrossRef] 50. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016,33, 1870–1874. [CrossRef] 51. Costa-Broseta, Á.; Perea-Resa, C.; Castillo, M.; Salinas, J.; León, J. Nitric oxide deficiency decreases C-repeat binding factordependent and -independent induction of cold acclimation. J. Exp. Bot. 2019, 3285–3296. [CrossRef] 52. Lata, C.; Prasad, M. Role of DREBs in regulation of abiotic stress responses in plants. J. Exp. Bot. 2011 ,62, 4731–4748. [CrossRef] 53. Shi, Y.; Ding, Y.; Yang, S. Molecular Regulation of CBF Signaling in Cold Acclimation. Trends Plant Sci. 2018 ,23, 623–637. [CrossRef] [PubMed] 54. Yahia, N.; Wani, S.H.; Kumar, V. CBF-Dependent and CBF-Independent transcriptional regulation of cold stress responses in plants. In Cold Tolerance in Plants; Wani, S.H., Herath, V., Eds.; Springer: Cham, Switzerland, 2018; pp. 89–102; ISBN 9783030014155. 55. Pons, C.; Martí, C.; Forment, J.; Crisosto, C.H.; Dandekar, A.M.; Granell, A. A bulk segregant gene expression analysis of a peach population reveals components of the underlying mechanism of the fruit cold response. PLoS ONE 2014,9, e90706. [CrossRef] 56. Zhang, Z.; Zhu, Q.; Hu, M.; Gao, Z.; An, F.; Li, M.; Jiang, Y. Low-temperature conditioning induces chilling tolerance in stored mango fruit. Food Chem. 2017,219, 76–84. [CrossRef] [PubMed] 57. Zhao, R.; Sheng, J.; Lv, S.; Zheng, Y.; Zhang, J.; Yu, M.; Shen, L. Nitric oxide participates in the regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit. Postharvest Biol. Technol. 2011,62, 121–126. [CrossRef] 58. Albornoz, K.; Cantwell, M.I.; Zhang, L.; Beckles, D.M. Integrative analysis of postharvest chilling injury in cherry tomato fruit reveals contrapuntal spatiotemporal responses to ripening and cold stress. Sci. Rep. 2019,9–2795, 1–14. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021,22, 804 20 of 20 59. Zacarias, L.; Cronje, P.J.R.; Palou, L. Postharvest technology of citrus fruits. In The Genus Citrus; Talon, M., Caruso, M., Gmitter, F.G., Eds.; Elsevier: Amsterdam, The Netherlands, 2020; pp. 421–446. ISBN 978-0-12-812163-4. 60. Curk, F.; Navarro, L. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Ann. Bot. 2016 ,117, 565–583. [CrossRef] [PubMed] 61. Canella, D.; Gilmour, S.J.; Kuhn, L.A.; Thomashow, M.F. Biochimica et Biophysica Acta DNA binding by the Arabidopsis CBF1 transcription factor requires the PKKP/RAGRxKFxETRHP signature sequence. Biochim. Biophys. Acta 2010 ,1799, 454–462. [CrossRef] 62. Lado, J.; Alós, E.; Rodrigo, M.J.; Zacarías, L. Light avoidance reduces ascorbic acid accumulation in the peel of Citrus fruit. Plant Sci. 2015,231, 138–147. [CrossRef] 63. Magwaza, L.S.; Opara, U.L.; Cronje, P.J.R.; Landahl, S.; Terry, L.A. Canopy position affects rind biochemical profile of “Nules Clementine” mandarin fruit during postharvest storage. Postharvest Biol. Technol. 2013,86, 300–308. [CrossRef] 64. Lado, J.; Alós, E.; Manzi, M.; Cronje, P.J.R.; Gómez-Cadenas, A.; Rodrigo, M.J.; Zacarías, L. Light Regulation of Carotenoid Biosynthesis in the Peel of Mandarin and Sweet Orange Fruits. Front. Plant Sci. 2019,10, 1–16. [CrossRef] 65. Pfaffl, M.W.; Horgan, G.W.; Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002,30, e36. [CrossRef] [PubMed] 66. Alós, E.; Rodrigo, M.J.; Zacarías, L. Differential transcriptional regulation of l-ascorbic acid content in peel and pulp of citrus fruits during development and maturation. Planta 2014,239, 1113–1128. [CrossRef] [PubMed]