Lorenzo Zacarías

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.

Molecular and functional characterization of a novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene accumulation

Carotenoids are the main pigments responsible of the colouration of Citrus fruits. The β-cyclization of lycopene, catalysed by the lycopene β-cyclases (β-LCY), seems to be a key regulatory step of the carotenoid pathway. In the present study, two β-LCYs from orange fruits (Citrus sinensis), named Csβ-LCY1 and Csβ-LCY2 have been isolated and the activity of the encoded proteins was demonstrated by functional analysis. Csβ-LCY1 was expressed at low levels and remained relatively constant during fruit ripening while Csβ-LCY2 showed a chromoplast-specific expression and a marked induction in both peel and pulp of orange fruits in parallel with the accumulation of β,β-xanthophylls. The potential involvement of Csβ-LCY2 in the accumulation of lycopene, characteristic of some Citrus species such as red grapefruits, was investigated. Expression of Csβ-LCY2 and another seven carotenoid biosynthetic genes were studied in the peel and pulp of the high lycopene-accumulating grapefruit, Star Ruby, and compared with those of ordinary Navel orange. In Star Ruby, the accumulation of lycopene during fruit maturation was associated with a substantial reduction in the expression of both β-LCY2 and β-CHX genes with respect to Navel orange. Moreover, two different alleles of β-LCY2: β-LCY2a and β-LCY2b were isolated from both genotypes, and functional assays demonstrated that the lycopene β-cyclase activity of the allele b was almost null. Interestingly, Star Ruby grapefruit predominantly expressed the unfunctional β-LCY2b allele during fruit ripening whereas Navel oranges preferably expressed the functional allele. It is suggested that the presence of diverse alleles of the β-LCY2 gene, encoding enzymes with altered activity, with different transcript accumulation may be an additional regulatory mechanism of carotenoid synthesis involved in the accumulation of lycopene in red grapefruits.

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.

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.

Terpene Down-Regulation in Orange Reveals the Role of Fruit Aromas in Mediating Interactions with Insect Herbivores and Pathogens

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.

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.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8′-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7′,8′ double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7′,8′ double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection.

BackgroundPostharvest losses of citrus fruit due to green mold decay, caused by the fungus Penicillium digitaum, have a considerable economic impact. However, little is known about the molecular processes underlying the response of citrus fruit to P. digitatum.ResultsHere we describe the construction of a subtracted cDNA library enriched in citrus genes preferentially expressed in response to pathogen infection followed by cDNA macroarray hybridization to investigate gene expression during the early stages of colonization of the fruits peel by P. digitatum. Sequence annotation of clones from the subtracted cDNA library revealed that induction of secondary and amino acid metabolisms constitutes the major response of citrus fruits to P. digitatum infection. Macroarray hybridization analysis was conducted with RNA from either control, wounded, ethylene treated or P. digitatum infected fruit. Results indicate an extensive overlap in the response triggered by the three treatments, but also demonstrated specific patterns of gene expression in response to each stimulus. Collectively our data indicate a significant presence of isoprenoid, alkaloid and phenylpropanoid biosynthetic genes in the transcriptomic response of citrus fruits to P. digitatum infection. About half of the genes that are up-regulated in response to pathogen infection are also induced by ethylene, but many examples of ethylene-independent gene regulation were also found. Two notable examples of this regulation pattern are the genes showing homology to a caffeine synthase and a berberine bridge enzyme, two proteins involved in alkaloid biosynthesis, which are among the most induced genes upon P. digitatum infection but are not responsive to ethylene.ConclusionsThis study provided the first global picture of the gene expression changes in citrus fruit in response to P. digitatum infection, emphasizing differences and commonalities with those triggered by wounding or exogenous ethylene treatment. Interpretation of the differentially expressed genes revealed that metabolism is redirected to the synthesis of isoprenes, alkaloids and phenylpropanoids.

Root ABA Accumulation in Long-Term Water-Stressed Plants is Sustained by Hormone Transport from Aerial Organs

The reduced pool of the ABA precursors, β,β-carotenoids, in roots does not account for the substantial increase in ABA content in response to water stress (WS) conditions, suggesting that ABA could be transported from other organs. Basipetal transport was interrupted by stem-girdling, and ABA levels were determined in roots after two cycles of WS induced by transplanting plants to dry perlite. Leaf applications of isotope-labeled ABA and reciprocal grafting of ABA-deficient tomato mutants were used to confirm the involvement of aerial organs on root ABA accumulation. Disruption of basipetal transport reduced ABA accumulation in roots, and this decrease was more severe after two consecutive WS periods. This effect was linked to a sharp decrease in the β,β-carotenoid pool in roots in response to water deficit. Significant levels of isotope-labeled ABA were transported from leaves to roots, mainly in plants subjected to water dehydration. Furthermore, the use of different ABA-deficient tomato mutants in reciprocal grafting combinations with wild-type genotypes confirmed the involvement of aerial organs in the ABA accumulation in roots. In conclusion, accumulation of ABA in roots after long-term WS periods largely relies on the aerial organs, suggesting a reduced ability of the roots to synthesize ABA from carotenoids. Furthermore, plants are able to transport ABA basipetally to sustain high hormone levels in roots.