Manuel Cercós

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

BackgroundImprovement 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.ResultsA 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 × 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 domainsConclusionComparative 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.

Ethylene-induced differential gene expression during abscission of citrus leaves

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission.

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.

Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves

BackgroundAbscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications.ResultsEfficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding.ConclusionThe LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus.

Molecular Physiology of Development and Quality of Citrus

Abstract Citrus is the most economically important fruit crop in the world. Citrus fruits are classified as hesperidiums, berries of very special organization characterized by a juicy pulp made of vesicles within segments. Besides the typical fruit components, citrus fruit contain many organic compounds necessary for human diet and an extraordinary number of metabolites displaying valuable properties for health. In citrus, the concept of fruit quality comprises several other aspects intimately related to human health apart from physical attributes and diet components. Citrus also possess a rare combination of intriguing biological characteristics including an unusual reproductive biology, a non‐climacteric fruit ripening and several specific tree‐traits. The combination of these characteristics suggests that the study of fruit growth regulation in citrus may reveal original mechanisms based on explicit molecular differences and on exclusive genes. Citrus is, therefore, an excellent model to study fruit quality because of its peculiar fruiting, singular biochemistry and economical relevance. In this chapter, the progress that has been carried out in the research on the molecular determinants related to development and fruit quality of citrus is reviewed. The review also intends to provide a physiological frame for the implementation of the information generated during the past years. Molecular background is provided on the current status of principal reproductive processes related to fruit quality mainly flowering, fruiting, ripening, and abscission. We also have focused on main characteristic secondary bioactive compounds, as major contributors of aroma and flavour and finally, on the abiotic stresses influencing development and fruit growth.

A serine carboxypeptidase gene (PsCP), expressed in early steps of reproductive and vegetative development in Pisum sativum, is induced by gibberellins

A cDNA clone encoding a serine carboxypeptidase (PsCP), isolated from young fruits of Pisum sativum L., was used to study the temporal and spatial expression and hormonal regulation of serine carboxypeptidase during reproductive and vegetative development. In unpollinated pea ovaries PsCP transcript levels decreased during senescence. However, during early fruit development, PsCP transcript were accumulated in both pericarp and seeds, preferentially in the nucellus, with a polar distribution at the chalazal region of the embryo sac, suggesting a role in seed development. PsCP transcript levels increased also when fruit set was induced in unpollinated ovaries by gibberellins, although the distribution was uniform. PsCP expression was also induced by auxins but not cytokinins, indicating a selective hormonal regulation of PsCP transcription. Localization of PsCP transcript after pollination parallel reported changes in gibberellin distribution, suggesting that PsCP transcription in developing fruits and seeds is induced by gibberellins. PsCP is also expressed in developing seedlings but not in cotyledons, suggesting that it is not involved in the mobilization of storage materials. PsCP transcripts were suppressed by treatment of seedlings with paclobutrazol and restored by gibberellic acid (GA3) treatment. In addition, PsCP transcript levels decreased in etiolated pea seedlings when they were exposed to continuous light but not when exposed to light in the presence of GA3. These results indicate that PsCP transcript accumulation is induced by gibberellins in developing seedlings. This is the first report of a serine carboxypeptidase-like gene induced by gibberellins in reproductive and vegetative developing tissues in dicotyledoneous plants.

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

BackgroundExternal 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.ResultsPigment 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 β-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.ConclusionsIn 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 β-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.

Early gene expression events in the laminar abscission zone of abscission-promoted citrus leaves after a cycle of water stress/rehydration: involvement of CitbHLH1

Leaf abscission is a common response of plants to drought stress. Some species, such as citrus, have evolved a specific behaviour in this respect, keeping their leaves attached to the plant body during water stress until this is released by irrigation or rain. This study successfully reproduced this phenomenon under controlled conditions (24h of water stress followed by 24h of rehydration) and used it to construct a suppression subtractive hybridization cDNA library enriched in genes involved in the early stages of rehydration-promoted leaf abscission after water stress. Sequencing of the library yielded 314 unigenes, which were spotted onto nylon membranes. Membrane hybridization with petiole (Pet)- and laminar abscission zone (LAZ)-enriched RNA samples corresponding to early steps in leaf abscission revealed an almost exclusive preferential gene expression programme in the LAZ. The data identified major processes such as protein metabolism, cell-wall modification, signalling, control of transcription and vesicle production, and transport as the main biological processes activated in LAZs during the early steps of rehydration-promoted leaf abscission after water stress. Based on these findings, a model for the early steps of citrus leaf abscission is proposed. In addition, it is suggested that CitbHLH1, the putative citrus orthologue of Arabidopsis BIGPETAL, may play major roles in the control of abscission-related events in citrus abscission zones.

Expression of cysteine proteinase mRNA in chickpea (Cicer arietinum L.) is localized to provascular cells in the developing root

Summary A full-length cDNA clone encoding a chickpea (Cicer arietinum L.) cysteine proteinase, homologous to rd21gene of Arabidopsis as well as to cysteine proteinase genes from legumes, was isolated. Expression of the mRNA in growing radicles has been located in a very precisely defined longitudinal zone in the stele, corresponding to the area of differentiation of vascular tissues. This result suggests that the encoded proteinase may be involved in programmed cell death events that precede vascular development.

Expression of PsGRP1, a novel glycine rich protein gene of Pisum sativum, is induced in developing fruit and seed and by ABA in pistil and root

A novel glycine-rich protein gene, PsGRP1, has been identified in Pisum sativum L. Accumulation of PsGRP1 transcripts was observed in reproductive organs and vegetative tissues. They were localized in endocarp sclerenchyma during fruit development in cells that will lignify. PsGRP1 expression was also detected in senescent pistils and developing seeds and induced by ABA treatment in presenescent pistils. A raise in the expression was also observed in roots after treatment with ABA or mannitol but not under cold stress. A mannitol treatment induced a rise in ABA levels and fluridone treatment counteracted the mannitol induction of PsGRP1 expression. The results suggest a possible role for PsGRP1 in differentiation of the endocarp sclerenchyma and during seed development, pistil senescence and osmotic stress under ABA control.