María T. Lafuente

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Catalase enzyme activity is related to tolerance of mandarin fruits to chilling

Abstract The effect of a postharvest hot-water dip treatment (HWT) at 53°C for 3 min and a 3-day heat-conditioning treatment at 37°C with air (HAT) at 90–95% RH on chilling tolerance and catalase (CAT) activity was compared in ‘Fortune’ mandarins. The HWT treatment increased CAT activity in the fruit, but after they were removed from high temperature to cold storage a rapid decline in CAT activity was associated with increased chilling injury. Greater chilling tolerance and CAT activity was induced when fruits were conditioned for 3 days at 37°C and 90–95% RH. The CAT activity in fruits exposed to HAT was higher than in the dipped and the non-heated fruits over the storage period at 2°C. An inhibitor of CAT activity, 3-amino-1,2,4-triazole (AT), caused peel damage in HAT ‘Fortune’ mandarins and in the chilling-tolerant ‘Clementine’ and ‘Clemenules’ cultivars stored at 2°C but not at 12°C (non-chilling temperature). CAT activity was reduced about two to three times by AT upon cold storage in the cultivars studied. Little difference was found in the activity of ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) between AT-treated and non-treated fruits. The data indicate that CAT may be a major antioxidant enzyme involved in the defence mechanism of mandarin fruits against chilling stress. Our results also suggest that the different effectiveness of the heat-conditioning treatments in increasing chilling tolerance of ‘Fortune’ mandarins may be related to induction of CAT activity during heating and on its persistence during cold storage.

Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the flavedo of fruit but responsive to cold and water stress in leaves.

A cDNA encoding a dehydrin was isolated from the flavedo of the chilling-sensitive Fortune mandarin fruit (Citrus clementina Hort. Ex Tanaka x Citrus reticulata Blanco) and designed as Crcor15. The predicted CrCOR15 protein is a K2S member of a closely related dehydrin family from Citrus, since it contains two tandem repeats of the unusual Citrus K-segment and one S-segment (serine cluster) at an unusual C-terminal position. Crcor15 mRNA is consistently and highly expressed in the flavedo during fruit development and maturation. The relative abundance of Crcor15 mRNA in the flavedo was estimated to be higher than 1% of total RNA. The high mRNA level remained unchanged during fruit storage at chilling (2 degrees C) and nonchilling (12 degrees C) temperatures, and it was depressed by a conditioning treatment (3 days at 37 degrees C) that induced chilling tolerance. Therefore, the expression of Crcor15 appears not to be related to the acquisition of chilling tolerance in mandarin fruits. However, Crcor15, which was barely detected in unstressed mandarin leaves, was rapidly induced in response to both low temperature and water stress. COR15 protein was expressed in Escherichia coli, and the purified protein conferred in vitro protection against freezing and dehydration inactivation. The potential role of Citrus COR15 is discussed.

Abscisic Acid in the Response of ‘Fortune’ Mandarins to Chilling. Effect of Maturity and High-Temperature Conditioning

The possible role of abscisic acid (ABA) and peel colour in chilling tolerance of ‘Fortune’ mandarins has been examined. Fruit stored at 2·5°C, but not at 12°C, showed pitting damage. The degree of damage changed considerably during development and maturity from November until May. Green fruit were more tolerant to chilling injury (CI). After colour break, chilling susceptibility increased from the beginning of December until January and February (the coolest season during the growing period) and declined towards May. This trend did not parallel changes in fruit colour index or free and bound ABA levels, which increased from November until May. At the time of chloroplast transformation to chromoplast a noticeable shift in free but not in bound ABA occurred. During storage, free ABA levels increased in fruits stored at 12°C, but not at 2·5°C. Changes in ABA with storage temperature depended on the stage of maturity of the fruits, but not on their susceptibility to CI. It has also been shown that there was no relationship between ABA content following exposure to 37°C and chilling tolerance. At all stages of maturity, the susceptibility of the fruits to CI and the free ABA levels of the flavedo decreased after conditioning the fruits for 3 days at 37°C. After storage at 2·5°C, the ABA levels of the conditioned fruit were similar or even lower than those of non-conditioned fruit. Treatment of fruit for 3 days with 10 μl litre-1 ethylene increased free ABA about four times, whereas bound ABA remained nearly constant. This treatment enhanced cold-induced peel damage after prolonged storage. Thus, the ethylene-induced changes in free ABA appear not to be related to the induced changes in chilling susceptibility. The results obtained indicated, therefore, that free and bound ABA appear not to be involved on the adaptative mechanism developed in ‘Fortune’ mandarins to cope with chilling stress.

PHENYLALANINE AMMONIA-LYASE AND ETHYLENE IN RELATION TO CHILLING INJURY AS AFFECTED BY FRUIT AGE IN CITRUS

Abstract Fruit of many citrus cultivars become injured when exposed to low, non-freezing temperatures. In this study we have determined changes in ethylene production and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in fruit of three citrus cultivars, ‘Fortune’ mandarins, and ‘Navelina’ and ‘Valencia’ late oranges, with different tolerance to chilling injury (CI) and demonstrated the influence of fruit physiological stage on those stress responses. We have shown that the increase in ethylene production and PAL are cold-induced responses which are only stimulated in fruit of citrus cultivars showing chilling damage and that both responses may occur concomitantly with the development of chilling symptoms. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. The influence of fruit age on both responses was evaluated in the most (‘Navelina’) and the least (‘Fortune’) chilling tolerant cultivars. Chilling damage was not developed in ‘Navelina’ fruit at any physiological stage, but our results in ‘Fortune’ mandarins, which always developed chilling symptoms, indicated that the induction of PAL in response to chilling was dependent on the fruit physiological stage. Interestingly, increases in both PAL mRNA and activity were barely affected by cold stress in the youngest ‘Fortune’ fruit harvested in December in spite of its noticeable CI. For a similar CI index, the older the fruit, the higher was the shift in the levels of PAL transcript and in PAL activity in response to cold. In contrast, the cold-induced ethylene production was little affected by the physiological stage of the fruit.

The Citrus ABA signalosome: identification and transcriptional regulation during sweet orange fruit ripening and leaf dehydration

The abscisic acid (ABA) signalling core in plants include the cytosolic ABA receptors (PYR/PYL/RCARs), the clade-A type 2C protein phosphatases (PP2CAs), and the subclass III SNF1-related protein kinases 2 (SnRK2s). The aim of this work was to identify these ABA perception system components in sweet orange and to determine the influence of endogenous ABA on their transcriptional regulation during fruit development and ripening, taking advantage of the comparative analysis between a wild-type and a fruit-specific ABA-deficient mutant. Transcriptional changes in the ABA signalosome during leaf dehydration were also studied. Six PYR/PYL/RCAR, five PP2CA, and two subclass III SnRK2 genes, homologous to those of Arabidopsis, were identified in the Citrus genome. The high degree of homology and conserved motifs for protein folding and for functional activity suggested that these Citrus proteins are bona fide core elements of ABA perception in orange. Opposite expression patterns of CsPYL4 and CsPYL5 and ABA accumulation were found during ripening, although there were few differences between varieties. In contrast, changes in expression of CsPP2CA genes during ripening paralleled those of ABA content and agreeed with the relevant differences between wild-type and mutant fruit transcript accumulation. CsSnRK2 gene expression continuously decreased with ripening and no remarkable differences were found between cultivars. Overall, dehydration had a minor effect on CsPYR/PYL/RCAR and CsSnRK2 expression in vegetative tissue, whereas CsABI1, CsAHG1, and CsAHG3 were highly induced by water stress. The global results suggest that responsiveness to ABA changes during citrus fruit ripening, and leaf dehydration was higher in the CsPP2CA gene negative regulators than in the other ABA signalosome components.

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and -independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.

Lignin and gum deposition in wounded ‘Oroval’ clementines as affected by chilling and peel water content

Abstract Induction of lignin synthesis and gum deposition by wounding in the albedo and flavedo of ‘Oroval’ clementines exposed to low temperature has been studied. Wounded and non-wounded fruit were kept for 24 h at 20 °C (initial separate experiment), six and 18 days at 2.5 °C (cold storage), and 18 days at 2.5 °C plus two additional days at 20 °C (shelf-life). At 2.5 °C, fruit were kept under two different conditions: saturated humidity or at 85–90% relative humidity (RH). During the separate initial control and shelf-life periods RH was 60%. The greatest weight loss (WL) was found in wounded fruit stored at 85–90% RH. Chilling injury (CI) was enhanced by wounding in all treatments. The most severe water loss occurred in flavedo of wounded fruit kept at 85–90% RH but, not surprisingly, at saturated humidity, no difference was found. Albedo water content was lower than that of flavedo and decreased again after shelf-life. Cell wall residue (CWR) progressively increased during cold storage in both albedo and flavedo of wounded fruit kept at 85–90% RH. Lignin-thioglycolic compounds increased in flavedo and albedo of wounded fruit kept at 20 °C during the 24-h period of the separate initial control. Under cold storage, however, these compounds did not increase together with CI development and fruit senescence. Wounding, CI and senescence considerably increased the deposition of ethanol-extractable phloroglucinol/HCl- (pg/HCl) positive compounds in flavedo, indicating (specific test) that wound gum deposition was more likely related than lignin biosynthesis to healing process and to pitting development under chilling conditions.

Ultrastructural and histochemical analysis reveals ethylene-induced responses underlying reduced peel collapse in detached citrus fruit

Fruits from many citrus cultivars develop depressed areas in the flavedo (outer part of the peel) and albedo (inner part) following detachment. Although ultrastructural analysis may provide important information about multiple plant responses to stresses and external stimuli at the cell and tissue levels, and despite the proved efficacy of ethylene in reducing peel damage in citrus fruit, cytological responses of this horticultural crop to protective ethylene concentrations have not yet been reported. We show that applying high ethylene levels (2 μL L−1 for 14 days) causes sublethal stress as it favored the alteration of cuticle, vacuole, middle lamella and primary wall, especially in the albedo cells, but reduced peel collapse in detached mature “Navelate” oranges (C. sinensis, L. Osbeck) held under nonstressful environmental conditions (22°C and 90–95% RH). Ethylene did not induce relevant changes in lignification but favored the deposition of pectic exudates and the release of sugars from degradation of cell polysaccharides including starch, cellulose, and pectins. In contrast, inhibiting ethylene perception by applying 1‐methylcyclopropene (1‐MCP) reduced these ethylene‐related responses and favored degradation of cell membranes and peel damage. The overall results reflect that mature oranges tolerate high ethylene levels that might favor the activation of defense responses involving oxidative‐stress related mechanisms and recycling of nutrients and carbon supply to enable cells to sustain respiration and cope with carbon deprivation stress caused by detachment. Microsc. Res. Tech. 2011.

LED Blue Light-induced changes in phenolics and ethylene in citrus fruit: Implication in elicited resistance against Penicillium digitatum infection

The objective was to investigate whether LED Blue Light (LBL) induces changes in phenolics and ethylene production of sweet oranges, and whether they participate in LBL-elicited resistance against the most important postharvest pathogen (Penicillium digitatum) of citrus fruit. The expression of relevant genes of the phenylpropanoid and ethylene biosynthetic pathways during elicitation of resistance was also determined. Different LBL (wavelength 450nm) quantum fluxes were used within the 60-630μmolm-2s-1 range. The HPLC analysis showed that the most relevant increase in phenylpropanoids occurred in scoparone, which markedly increased 3days after exposing fruits to a very high quantum flux (630μmolm-2s-1) for 18h. However, phenylpropanoids, including scoparone, were not critical factors in LBL-induced resistance. The genes involved in ethylene biosynthesis were differentially regulated by LBL. Ethylene is not involved in elicited resistance, although high LBL levels increased ethylene production in only 1h.