Ascorbic Acid Content and Transcriptional Profiling of Genes Involved in Its Metabolism during Development of Petals, Leaves, and Fruits of Orange (Citrus sinensis cv. Valencia Late).

Citrus fruit is one of the most important contributors to the ascorbic acid (AsA) intake in humans. Here, we report a comparative analysis of AsA content and transcriptional changes of genes related to its metabolism during development of petals, leaves and fruits of Valencia Late oranges (Citrus sinensis). Petals of close flowers and at anthesis contained the highest concentration of AsA. In fruits, AsA content in the flavedo reached a maximum at color break, whereas the pulp accumulated lower levels and experienced minor fluctuations during development. AsA levels in leaves were similar to those in the flavedo at breaker stage. The transcriptional profiling of AsA biosynthetic, degradation, and recycling genes revealed a complex and specific interplay of the different pathways for each tissue. The D-galacturonic acid pathway appeared to be relevant in petals, whereas in leaves the L-galactose pathway (GGP and GME) also contributed to AsA accumulation. In the flavedo, AsA content was positively correlated with the expression of GGP of the L-galactose pathway and negatively with DHAR1 gene of the recycling pathway. In the pulp, AsA appeared to be mainly controlled by the coordination among the D-galacturonic acid pathway and the MIOX and GalDH genes. Analysis of the promoters of AsA metabolism genes revealed a number of cis-acting elements related to developmental signals, but their functionalities remain to be investigated.

Dynamics of ascorbic acid content in apple (Malus x domestica) during fruit development and storage.

Vitamin C is a crucial antioxidant and cofactor for both plants and humans. Apple fruits generally contain low levels of vitamin C, making vitamin C content an interesting trait for apple crop improvement. With the aim of breeding high vitamin C apple cultivars it is important to get an insight in the natural biodiversity of vitamin C content in apple fruits. In this study, quantification of ascorbic acid (AsA), dehydroascorbic acid (DHA), and total AsA (AsA + DHA) in apple pulp of 79 apple accessions at harvest revealed significant variation, indicating a large genetic biodiversity. High density genotyping using an 8 K SNP array identified 21 elite and 58 local cultivars in this germplasm, with local accessions showing similar levels of total AsA but higher amounts of DHA compared to elite varieties. Out of the 79 apple cultivars screened, ten genotypes with either the highest or the lowest concentration of total AsA at harvest were used for monitoring vitamin C dynamics during fruit development and storage. For all these cultivars, the AsA/DHA ratio in both apple pulp and peel increased throughout fruit development, whereas the AsA/DHA balance always shifted towards the oxidized form during storage and shelf life, putatively reflecting an abiotic stress response. Importantly, at any point during apple fruit development and storage, the apple peel contained a higher level of vitamin C compared to the pulp, most likely because of its direct exposure to abiotic and biotic stresses.

A mutant allele of ζ-carotene isomerase (Z-ISO) is associated with the yellow pigmentation of the "Pinalate" sweet orange mutant and reveals new insights into its role in fruit carotenogenesis.

BACKGROUND: Fruit coloration is one of the main quality parameters of Citrus fruit primarily determined by genetic factors. The fruit of ordinary sweet orange (Citrus sinensis) displays a pleasant orange tint due to accumulation of carotenoids, representing ß,ß-xanthophylls more than 80% of the total content. 'Pinalate' is a spontaneous bud mutant, or somatic mutation, derived from sweet orange 'Navelate', characterized by yellow fruits due to elevated proportions of upstream carotenes and reduced ß,ß-xanthophylls, which suggests a biosynthetic blockage at early steps of the carotenoid pathway. RESULTS: To identify the molecular basis of 'Pinalate' yellow fruit, a complete characterization of carotenoids profile together with transcriptional changes in carotenoid biosynthetic genes were performed in mutant and parental fruits during development and ripening. 'Pinalate' fruit showed a distinctive carotenoid profile at all ripening stages, accumulating phytoene, phytofluene and unusual proportions of 9,15,9'-tri-cis- and 9,9'-di-cis-ζ-carotene, while content of downstream carotenoids was significantly decreased. Transcript levels for most of the carotenoid biosynthetic genes showed no alterations in 'Pinalate'; however, the steady-state level mRNA of ζ-carotene isomerase (Z-ISO), which catalyses the conversion of 9,15,9'-tri-cis- to 9,9'-di-cis-ζ-carotene, was significantly reduced both in 'Pinalate' fruit and leaf tissues. Isolation of the 'Pinalate' Z-ISO genomic sequence identified a new allele with a single nucleotide insertion at the second exon, which generates an alternative splicing site that alters Z-ISO transcripts encoding non-functional enzyme. Moreover, functional assays of citrus Z-ISO in E.coli showed that light is able to enhance a non-enzymatic isomerization of tri-cis to di-cis-ζ-carotene, which is in agreement with the partial rescue of mutant phenotype when 'Pinalate' fruits are highly exposed to light during ripening. CONCLUSION: A single nucleotide insertion has been identified in 'Pinalate' Z-ISO gene that results in truncated proteins. This causes a bottleneck in the carotenoid pathway with an unbalanced content of carotenes upstream to ß,ß-xanthophylls in fruit tissues. In chloroplastic tissues, the effects of Z-ISO alteration are mainly manifested as a reduction in total carotenoid content. Taken together, our results indicate that the spontaneous single nucleotide insertion in Z-ISO is the molecular basis of the yellow pigmentation in 'Pinalate' sweet orange and points this isomerase as an essential activity for carotenogenesis in citrus fruits.

Light Regulation of Carotenoid Biosynthesis in the Peel of Mandarin and Sweet Orange Fruits.

Carotenoids are the pigments responsible for the coloration of the peel and pulp of Citrus fruits. Light is one of the major environmental factors influencing coloration and carotenoid content and composition of fleshy fruits and therefore their commercial and nutritional quality. Agronomical observations indicate that citrus fruits exposed to sunlight develop a brighter peel coloration than shaded fruit inside the tree canopy. In the present study, the effect of light deprivation on carotenoid profile, and in the expression of genes of carotenoid metabolism and their precursors have been analyzed in fruits of Clemenules mandarin (Citrus clementine) and Navelina orange (Citrus sinensis). Fruit shading accelerated peel degreening, chlorophyll degradation, and reduced chloroplastic-type carotenoids. Time-course shading experiments revealed that the stage of fruit ripening appears to be determinant for the effect of darkness in carotenoid biosynthesis. Fruit shading produced a down-regulation of the expression of key carotenoids biosynthetic genes (PSY, PDS, ZDS1, LCY2a, LCY2b, and CHX). However, expression of MEP pathway genes (DXS, HDR1, and GGPPS1) and the carotenoid cleavage dioxygenase, CCD4b1, responsible of the formation of the apocarotenoid ß-citraurin, were not substantially affected by dark-grown conditions. The content of abscisic acid (ABA), an end product of the carotenoid pathway, was not affected by the light regime, suggesting that effect of shading on the precursor's pool is not sufficient to impair ABA synthesis. A moderate increase in total carotenoid and in the expression of biosynthetic genes was observed in mature dark-grown mandarin and orange fruits. Collectively, results suggest that light stimulates carotenoid biosynthesis in the peel of citrus fruits but a light-independent regulation may also operate.

Manipulation of Carotenoid Content in Plants to Improve Human Health.

Carotenoids are essential components for human nutrition and health, mainly due to their antioxidant and pro-vitamin A activity. Foods with enhanced carotenoid content and composition are essential to ensure carotenoid feasibility in malnourished population of many countries around the world, which is critical to alleviate vitamin A deficiency and other health-related disorders. The pathway of carotenoid biosynthesis is currently well understood, key steps of the pathways in different plant species have been characterized and the corresponding genes identified, as well as other regulatory elements. This enables the manipulation and improvement of carotenoid content and composition in order to control the nutritional value of a number of agronomical important staple crops. Biotechnological and genetic engineering-based strategies to manipulate carotenoid metabolism have been successfully implemented in many crops, with Golden rice as the most relevant example of ß-carotene improvement in one of the more widely consumed foods. Conventional breeding strategies have been also adopted in the bio-fortification of carotenoid in staple foods that are highly consumed in developing countries, including maize, cassava and sweet potatoes, to alleviate nutrition-related problems. The objective of the chapter is to summarize major breakthroughs and advances in the enhancement of carotenoid content and composition in agronomical and nutritional important crops, with special emphasis to their potential impact and benefits in human nutrition and health.

Pseudomonas fluorescens PICF7 displays an endophytic lifestyle in cultivated cereals and enhances yield in barley.

Pseudomonas fluorescens PICF7, an indigenous inhabitant of olive roots, displays an endophytic lifestyle in this woody crop and exerts biocontrol against the fungal phytopathogen Verticillium dahliae Here we report microscopy evidence that the strain PICF7 is also able to colonize and persist on or in wheat and barley root tissues. Root colonization of both cereal species followed a similar pattern to that previously reported in olive, including inner colonization of the root hairs. This demonstrates that strain PICF7 can colonize root systems of distant botanical species. Barley plants germinated from PICF7-treated seeds showed enhanced vegetative growth. Moreover, significant increases in the number of grains (up to 19.5%) and grain weight (up to 20.5%) per plant were scored in this species. In contrast, growth and yield were not significantly affected in wheat plants by the presence of PICF7. Proteomics analysis of the root systems revealed that different proteins were exclusively found depending on the presence or absence of PICF7 and only one protein with hydrogen ion transmembrane transporter activity was exclusively found in both PICF7-inoculated barley and wheat plants but not in the controls.

Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background.

The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7Hch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7Hchα and 7Hchß chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7Hchα∙7AL and 7AS∙7Hchß disomic translocation lines. The carotenoid content in 7Hchα∙7AL and 7AS∙7Hchß disomic translocation lines was higher than the wheat-7Hch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7Hch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7Hchα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds.

Effect of 7H(ch) Hordeum chilense chromosome introgressions on the wheat endosperm proteomic profile.

Hordeum chilense is an excellent genetic resource for wheat breeding due to its potential to improve breadmaking quality and nutritional value and provide resistance to some biotic and abiotic stresses. Hexaploid wheat lines carrying chromosome 7H(ch) introgressions, namely, chromosome additions of the whole chromosome 7H(ch) or the 7H(ch)α or the 7H(ch)ß chromosome arms, and chromosome substitutions of the homeologous chromosomes 7A, 7B, or 7D by chromosome 7H(ch) were compared by 2D-PAGE analysis to study the effect of these alien introgressions on the wheat endosperm proteome. The addition of the 7H(ch)α chromosome arm did not alter the profile of most glutenins and gliadins, but showed higher quantities of puroindolines and lower xylanase inhibitors, which might improve also resistance to plant pathogens. On the other hand, (7A)7H(ch) or (7D)7H(ch) substitution lines showed enhanced avenin-like b proteins and triticin levels but reduced puroindolines, which could be desirable to improve dough properties and nutritional value and increase kernel hardness in wheat.

Light avoidance reduces ascorbic acid accumulation in the peel of Citrus fruit.

Citrus fruits are highly consumed worldwide and represent one of the most important sources of ascorbic acid (AsA). However, information about the molecular mechanisms regulating AsA accumulation in Citrus fruit and the effects of environmental factors is scarce. In this study we have investigated the effect of fruit shading on AsA content and the expression of AsA biosynthetic, degrading and recycling genes in fruits of different Citrus species. Immature-green fruits were covered at the end of the cell enlargement phase and AsA concentration in the flavedo declined and remained at low levels as compared with light-exposed fruits. Fruit shading marginally altered the expression of genes from the l-galactose pathway and this effect was variable in the four Citrus species. However, specific isoforms (GalUR8 or GalUR12) from the l-galacturonic acid pathway were significantly repressed paralleling the reduction in AsA concentration. No significant effect of shading was detected in transcription of genes of the myo-inositol and l-gulose pathways as well as recycling and degradation. Collectively, results indicate that light avoidance inhibited accumulation of AsA in the flavedo of Citrus fruits and suggest that the l-galacturonic acid pathway has a relevant contribution to AsA content in this tissue.

Unravelling the proteomic profile of rice meiocytes during early meiosis.

Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

Differential transcriptional regulation of L-ascorbic acid content in peel and pulp of citrus fruits during development and maturation.

Citrus fruits are an important source of ascorbic acid (AsA) for human nutrition, but the main pathways involved in its biosynthesis and their regulation are still not fully characterized. To study the transcriptional regulation of AsA accumulation, expression levels of 13 genes involved in AsA biosynthesis, 5 in recycling and 5 in degradation were analyzed in peel and pulp of fruit of two varieties with different AsA concentration: Navel orange (Citrus sinensis) and Satsuma mandarin (Citrus unshiu). AsA accumulation in peel and pulp correlated with the transcriptional profiling of the L-galactose pathway genes, and the myo-inositol pathway appeared to be also relevant in the peel of immature-green orange. Differences in AsA content between varieties were associated with differential gene expression of GDP-mannose pyrophosphorylase (GMP), GDP-L-galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP), myo-inositol oxygenase in peel, and GGP and GPP in pulp. Relative expressions of monodehydroascorbate reductase 3 (MDHAR3) and dehydroascorbate reductase1 (DHAR1) correlated with AsA accumulation during development and ripening in peel and pulp, respectively, and were more highly expressed in the variety with higher AsA contents. Collectively, results indicated a differential regulation of AsA concentration in peel and pulp of citrus fruits that may change during the different stages of fruit development. The L-galactose pathway appears to be predominant in both tissues, but AsA concentration is regulated by complex mechanisms in which degradation and recycling also play important roles.

Altered sensitivity to ethylene in 'Tardivo', a late-ripening mutant of Clementine mandarin.

'Tardivo' mandarin is a mutant of 'Comune' Clementine with a delay in peel degreening and coloration, allowing late harvesting. In this work, we have explored if the late-harvesting phenotype of 'Tardivo' mandarin is related to altered perception and sensitivity to ethylene. The peel degreening rate was examined after a single ethephon treatment or during a continuous ethylene application in fruits at two maturation stages. In general, ethylene-induced peel degreening was considerably delayed and reduced in fruits of 'Tardivo', as well as the concomitant reduction of chlorophyll (Chl) and chloroplastic carotenoids, and the accumulation of chromoplastic carotenoids. Analysis of the expression of genes involved in Chl degradation, carotenoids, ABA, phenylpropanoids and ethylene biosynthesis revealed an impairment in the stimulation of most genes by ethylene in the peel of 'Tardivo' fruits with respect to 'Comune', especially after 5 days of ethylene application. Moreover, ethylene-induced expression of two ethylene receptor genes, ETR1 and ETR2, was also reduced in mutant fruits. Expression levels of two ethylene-responsive factors, ERF1 and ERF2, which were repressed by ethylene, were also impaired to a different extent, in fruits of both genotypes. Collectively, results suggested an altered sensitivity of the peel of 'Tardivo' to ethylene-induced physiological and molecular responses, including fruit degreening and coloration processes, which may be time-dependent since an early moderated reduction in the responses was followed by the latter inability to sustain ethylene action. These results support the involvement of ethylene in the regulation of at least some aspects of peel maturation in the non-climacteric citrus 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 ß-citraurin (3-hydroxy-ß-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 ß-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ß-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ß-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and ß-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.

Transcriptomic analysis of genes involved in the biosynthesis, recycling and degradation of L-ascorbic acid in pepper fruits (Capsicum annuum L.).

Sweet pepper (Capsicum annuum L.) is widely recognized among the vegetables with high content of ascorbic acid (AsA). However, the metabolic pathways involved in the biosynthesis, recycling and degradation of AsA and their relative contribution to the concentration of AsA have not been established yet. In the present work, the expression levels of selected genes involved in the AsA biosynthesis, degradation and recycling pathways were analyzed during development and ripening of pepper fruit cv. Palermo and in mature fruit of four cultivars (Lipari, C-116, Surrentino and Italverde) with different AsA concentrations. An inverse correlation was found between the expression of the biosynthetic genes and AsA concentrations, which could indicate that a feedback mechanism regulates AsA homeostasis in pepper fruits. Interestingly, analysis of mRNA levels of ascorbate oxidase, involved in the degradation of AsA, suggests that this enzyme plays a critical role in the regulation of the AsA pool during fruit development and ripening.

Transcription factor PIF4 controls the thermosensory activation of flowering.

Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change.

Identification of a GCC transcription factor responding to fruit colour change events in citrus through the transcriptomic analyses of two mutants.

BACKGROUND: External ripening in Citrus fruits is morphologically characterized by a colour shift from green to orange due to the degradation of chlorophylls and the accumulation of carotenoid pigments. Although numerous genes coding for enzymes involved in such biochemical pathways have been identified, the molecular control of this process has been scarcely studied. In this work we used the Citrus clementina mutants 39B3 and 39E7, showing delayed colour break, to isolate genes potentially related to the regulation of peel ripening and its physiological or biochemical effects. RESULTS: Pigment analyses revealed different profiles of carotenoid and chlorophyll modification in 39B3 and 39E7 mutants. Flavedo from 39B3 fruits showed an overall delay in carotenoid accumulation and chlorophyll degradation, while the flavedo of 39E7 was devoid of the apocarotenoid ß-citraurin among other carotenoid alterations. A Citrus microarray containing about 20,000 cDNA fragments was used to identify genes that were differentially expressed during colour change in the flavedo of 39B3 and 39E7 mutants respect to the parental variety. The results highlighted 73 and 90 genes that were respectively up- and down-regulated in both mutants. CcGCC1 gene, coding for a GCC type transcriptional factor, was found to be down-regulated. CcGCC1 expression was strongly induced at the onset of colour change in the flavedo of parental clementine fruit. Moreover, treatment of fruits with gibberellins, a retardant of external ripening, delayed both colour break and CcGCC1 overexpression. CONCLUSIONS: In this work, the citrus fruit ripening mutants 39B3 and 39E7 have been characterized at the phenotypic, biochemical and transcriptomic level. A defective synthesis of the apocarotenoid ß-citraurin has been proposed to cause the yellowish colour of fully ripe 39E7 flavedo. The analyses of the mutant transcriptomes revealed that colour change during peel ripening was strongly associated with a major mobilization of mineral elements and with other previously known metabolic and photosynthetic changes. The expression of CcGCC1 was associated with peel ripening since CcGCC1 down-regulation correlated with a delay in colour break induced by genetic, developmental and hormonal causes.

An evaluation of the basis and consequences of a stay-green mutation in the navel negra citrus mutant using transcriptomic and proteomic profiling and metabolite analysis.

A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown-colored flavedo during ripening. Analysis of pigment composition in the wild-type and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis, was impaired in the mutant, identifying nan as a type C stay-green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes and chlorophyllase activity but no accumulation of dephytylated Chl compounds during ripening, suggesting that the mutation is not related to a lesion in any of the principal enzymatic steps in Chl catabolism. Transcript profiling using a citrus microarray indicated that a citrus ortholog of a number of SGR (for STAY-GREEN) genes was expressed at substantially lower levels in nan, both prior to and during ripening. However, the pattern of catabolite accumulation and SGR sequence analysis suggested that the nan mutation is distinct from those in previously described stay-green mutants and is associated with an upstream regulatory step, rather than directly influencing a specific component of Chl catabolism. Transcriptomic and comparative proteomic profiling further indicated that the nan mutation resulted in the suppressed expression of numerous photosynthesis-related genes and in the induction of genes that are associated with oxidative stress. These data, along with metabolite analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate for the elevated Chl levels and associated photooxidative stress.

Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance.

BACKGROUND: Improvement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity. RESULTS: A normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis x Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domains CONCLUSION: Comparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays.

Regulation of color break in citrus fruits. Changes in pigment profiling and gene expression induced by gibberellins and nitrate, two ripening retardants.

Citrus clementina fruits were repeatedly treated on-tree from mature green until breaker stages with either nitrate or gibberellin, two retardants of external ripening. The natural color break was characterized by a reduction in chlorophyll concentration, a decrease in beta,epsilon-carotenoids, beta-carotene, neoxanthin, and all-E-violaxanthin, and an increase in beta,beta-xanthophylls [mainly (9Z)-violaxanthin and beta-cryptoxanthin]. The two retardants delayed both chlorophyll depletion and total carotenoid accumulation and in addition altered carotenoid composition. Treated fruits maintained longer the typical carotenoid composition of green fruits and reduced beta,beta-xanthophyll accumulation. Natural degreening was accompanied by a marked decrease in transcript levels of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and geranylgeranyl reductase (CHL P) while, conversely, pheophorbide a oxygenase (PaO) and phytoene synthase (PSY) gene expression increased. Gibberellin and nitrate delayed the reduction of DXS expression and the induction of PaO and PSY transcript accumulation, while no differences in CHL P were observed. The data indicate that both ripening retardants repressed natural PaO and PSY expression, suggesting a mechanistic basis for the elevated levels of chlorophyll and lower carotenoid concentration resulting from the gibberellin and nitrogen treatments and the consequent color break delay in citrus fruit peels.