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

<p>Abstract</p> <p>Background</p> <p>Abscission 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.</p> <p>Results</p> <p>Efficient 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 <it>in-vitro </it>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.</p> <p>Conclusion</p> <p>The 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.</p

An RNA-Seq-based reference transcriptome for Citrus

Previous RNA-Seq studies in citrus have been focused on physiological processes relevant to fruit quality and productivity of the major species, especially sweet orange. Less attention has been paid to vegetative or reproductive tissues, while most Citrus species have never been analysed. In this work, we characterized the transcriptome of vegetative and reproductive tissues from 12 Citrus species from all main phylogenetic groups. Our aims were to acquire a complete view of the citrus transcriptome landscape, to improve previous functional annotations and to obtain genetic markers associated with genes of agronomic interest. 28 samples were used for RNA-Seq analysis, obtained from 12 Citrus species: C.medica, C.aurantifolia, C.limon, C.bergamia, C.clementina, C.deliciosa, C.reshni, C.maxima, C.paradisi, C.aurantium, C.sinensis and Poncirus trifoliata. Four different organs were analysed: root, phloem, leaf and flower. A total of 3421 million Illumina reads were produced and mapped against the reference C.clementina genome sequence. Transcript discovery pipeline revealed 3326 new genes, the number of genes with alternative splicing was increased to 19739, and a total of 73797 transcripts were identified. Differential expression studies between the four tissues showed that gene expression is overall related to the physiological function of the specific organs above any other variable. Variants discovery analysis revealed the presence of indels and SNPs in genes associated with fruit quality and productivity. Pivotal pathways in citrus such as those of flavonoids, flavonols, ethylene and auxin were also analysed in detail

Prediction of components of the sporopollenin synthesis pathway in peach by genomic and expression analyses

Background: The outer cell wall of the pollen grain (exine) is an extremely resistant structure containing sporopollenin, a mixed polymer made up of fatty acids and phenolic compounds. The synthesis of sporopollenin in the tapetal cells and its proper deposition on the pollen surface are essential for the development of viable pollen. The beginning of microsporogenesis and pollen maturation in perennial plants from temperate climates, such as peach, is conditioned by the duration of flower bud dormancy. In order to identify putative genes involved in these processes, we analyzed the results of previous genomic experiments studying the dormancy-dependent gene expression in different peach cultivars. Results: The expression of 50 genes induced in flower buds after the endodormancy period (flower-bud late genes) was compared in ten cultivars of peach with different dormancy behaviour. We found two co-expression clusters enriched in putative orthologs of sporopollenin synthesis and deposition factors in Arabidopsis. Flower-bud late genes were transiently expressed in anthers coincidently with microsporogenesis and pollen maturation processes. We postulated the participation of some flower-bud late genes in the sporopollenin synthesis pathway and the transcriptional regulation of late anther development in peach. Conclusions: Peach and the model plant Arabidopsis thaliana show multiple elements in common within the essential sporopollenin synthesis pathway and gene expression regulatory mechanisms affecting anther development. The transcriptomic analysis of dormancy-released flower buds proved to be an efficient procedure for the identification of anther and pollen development genes in perennial plants showing seasonal dormancy

Transcriptome analysis of the pulp of citrus fruitlets suggests that domestication enhanced growth processes and reduced chemical defenses increasing palatability

To identify key traits brought about by citrus domestication, we have analyzed the transcriptomes of the pulp of developing fruitlets of inedible wild Ichang papeda (Citrus ichangensis), acidic Sun Chu Sha Kat mandarin (C. reticulata) and three palatable segregants of a cross between commercial Clementine (C. x clementina) and W. Murcott (C. x reticulata) mandarins, two pummelo/mandarin admixtures of worldwide distribution. RNA-seq comparison between the wild citrus and the ancestral sour mandarin identified 7267 differentially expressed genes, out of which 2342 were mapped to 117 KEGG pathways. From the remaining genes, a set of 2832 genes was functionally annotated and grouped into 45 user-defined categories. The data suggest that domestication promoted fundamental growth processes to the detriment of the production of chemical defenses, namely, alkaloids, terpenoids, phenylpropanoids, flavonoids, glucosinolates and cyanogenic glucosides. In the papeda, the generation of energy to support a more active secondary metabolism appears to be dependent upon upregulation of glycolysis, fatty acid degradation, Calvin cycle, oxidative phosphorylation, and ATP-citrate lyase and GABA pathways. In the acidic mandarin, downregulation of cytosolic citrate degradation was concomitant with vacuolar citrate accumulation. These changes affected nitrogen and carbon allocation in both species leading to major differences in organoleptic properties since the reduction of unpleasant secondary metabolites increases palatability while acidity reduces acceptability. The comparison between the segregants and the acidic mandarin identified 357 transcripts characterized by the occurrence in the three segregants of additional downregulation of secondary metabolites and basic structural cell wall components. The segregants also showed upregulation of genes involved in the synthesis of methyl anthranilate and furaneol, key substances of pleasant fruity aroma and flavor, and of sugar transporters relevant for sugar accumulation. Transcriptome and qPCR analysis in developing and ripe fruit of a set of genes previously associated with citric acid accumulation, demonstrated that lower acidity is linked to downregulation of these regulatory genes in the segregants. The results suggest that the transition of inedible papeda to sour mandarin implicated drastic gene expression reprograming of pivotal pathways of the primary and secondary metabolism, while palatable mandarins evolved through progressive refining of palatability properties, especially acidity

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

[EN] 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 zonesWork at the Centre de Genomica was supported by INIA grant RTA08-00065-00-00 and Ministerio de Ciencia e Innovacion-FEDER grants AGL2007-65437-C04-01, PSG-06-0000-2009-8, IPT-01-0000-2010-43, and AGL2011-30240. J.A. and P. M. were recipients of INIA predoctoral fellowships and M. C. and A. C. of INIA/CCAA and 'Ramon y Cajal' postdoctoral contracts, respectively. The help and expertise of E. Blazquez, I. Sanchis, and A. Boix are gratefully acknowledged.Agustí, J.; Gimeno, J.; Merelo, P.; Serrano Salom, R.; Cercós, M.; Conesa, A.; Talón, M.... (2012). Early gene expression events in the laminar abscission zone of abscission-promoted citrus leaves after a cycleof water stress/rehydration: involvement of CitbHLH1. Journal of Experimental Botany. 63:6079-6091. https://doi.org/10.1093/jxb/ers270S607960916

Comparative transcriptome analysis of stylar canal cells identifies novel candidate genes implicated in the self-incompatibility response of Citrus clementina

<p>Abstract</p> <p>Background</p> <p>Reproductive biology in citrus is still poorly understood. Although in recent years several efforts have been made to study pollen-pistil interaction and self-incompatibility, little information is available about the molecular mechanisms regulating these processes. Here we report the identification of candidate genes involved in pollen-pistil interaction and self-incompatibility in clementine (<it>Citrus clementina </it>Hort. ex Tan.). These genes have been identified comparing the transcriptomes of laser-microdissected stylar canal cells (SCC) isolated from two genotypes differing for self-incompatibility response ('Comune', a self-incompatible cultivar and 'Monreal', a self- compatible mutation of 'Comune').</p> <p>Results</p> <p>The transcriptome profiling of SCC indicated that the differential regulation of few specific, mostly uncharacterized transcripts is associated with the breakdown of self-incompatibility in 'Monreal'. Among them, a novel F-box gene showed a drastic up-regulation both in laser microdissected stylar canal cells and in self-pollinated whole styles with stigmas of 'Comune' in concomitance with the arrest of pollen tube growth. Moreover, we identify a non-characterized gene family as closely associated to the self-incompatibility genetic program activated in 'Comune'. Three different aspartic-acid rich (Asp-rich) protein genes, located in tandem in the clementine genome, were over-represented in the transcriptome of 'Comune'. These genes are tightly linked to a DELLA gene, previously found to be up-regulated in the self-incompatible genotype during pollen-pistil interaction.</p> <p>Conclusion</p> <p>The highly specific transcriptome survey of the stylar canal cells identified novel genes which have not been previously associated with self-pollen rejection in citrus and in other plant species. Bioinformatic and transcriptional analyses suggested that the mutation leading to self-compatibility in 'Monreal' affected the expression of non-homologous genes located in a restricted genome region. Also, we hypothesize that the Asp-rich protein genes may act as Ca²⁺"entrapping" proteins, potentially regulating Ca²⁺homeostasis during self-pollen recognition.</p

The LOV Protein of Xanthomonas citri subsp. citri Plays a Significant Role in the Counteraction of Plant Immune Responses during Citrus Canker

Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.Fil: Kraiselburd, Ivana. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Daurelio, Lucas Damian. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Tondo, Maria Laura. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Merelo, Paz. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Cortadi, Adriana Amalia. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Talón, Manuel. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Tadeo, Francisco R.. INSTITUT VALENCIÀ D'INVESTIGACIONS AGRÀRIES (IVIA);Fil: Orellano, Elena Graciela. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario

Histología y Citología de Cítricos

El cultivo de los cítricos es una tradición muy arraigada en toda la cuenca mediterránea. Esta práctica supone una fuente apreciable de riqueza para los habitantes de las comarcas o regiones que la explotan comercialmente. El estudio de los cítricos en España comenzó a principios del siglo pasado en torno a la Granja Agrícola de Burjassot, concretamente en la Estación Naranjera de Levante, una institución pionera constituida en el año 1931 y dedicada íntegramente a tal fin. Con posterioridad, a mitad de los años 70, el personal de la Estación se trasladó a otras instalaciones más modernas y espaciosas, localizadas en Montcada, que hoy conocemos como Instituto Valenciano de Investigaciones Agrarias (IVIA). En este Instituto se continúa profundizando hoy día en el conocimiento y en la mejora del cultivo de los cítricos. Los trabajos de histología y citología interesaron al Dr. Eduardo Primo Millo desde su inicio en el mundo de la investigación agraria. Suya fue la idea de, con el paso del tiempo, recopilar las aportaciones que su equipo venía realizando en este campo. Una antigua iniciativa que hoy se materializa en esta "Histología y Citología de Cítricos". Este texto comienza con la descripción tanto de las estructuras y de los sistemas membranosos que pueden contener las células como de los diferentes tejidos que componen un cítrico. A continuación se aborda la germinación de la semilla, un complejo proceso de degradación y movilización de reservas nutritivas que permitirán el crecimiento de estructuras vegetativas vitales para la planta: el tallo y la raíz. El estudio de la porción enterrada de la planta, la raíz, comienza con su ontogenia y su estructura primaria y prosigue incidiendo sobre su crecimiento secundario y su ramificación, así como sobre el papel que algunas fitohormonas pueden tener en estos procesos. La descripción del tallo, en particular de su sistema vascular, y de la hoja, se aborda haciendo hincapié en los cambios que experimenta la anatomía foliar en condiciones medioambientales que inducen estrés en los cítricos, como por ejemplo la salinidad. Para finalizar se describe la morfología y la anatomía de las estructuras reproductivas de los cítricos repasando los cambios anatómicos y ultraestructurales que se producen en las distintas partes de la flor y en el fruto durante los procesos de floración y de fructificación. La mayoría de las descripciones anatómicas, histológicas y ultraestructurales que aparecen en este texto están ilustradas con imágenes obtenidas en la investigación realizada en el Laboratorio de Fisiología Vegetal del Departamento de Citricultura y Otros Frutales del IVIA

Engineering of gibberellin levels in citrus by sense and antisense

http://jxb.oxfordjournals.org/Carrizo citrange (Citrus sinensis x Poncirus trifoliata) is a citrus hybrid 4 widely used as a rootstock, whose genetic manipulation to improve 5 different growth characteristics is of high agronomic interest. In this work 6 we have produced transgenic Carrizo citrange plants overexpressing 7 sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) 8 under control of the 35S promoter to modify plant architecture. As 9 expected, taller (sense) and shorter (antisense) phenotype correlated with 10 higher and lower levels, respectively, of active GA1 in growing shoots. In 11 contrast, other phenotypic characteristics seemed to be specific of citrus, 12 or different to those described for similar transgenics in other species. For 13 instance thorns, typical organs of citrus at juvenile stages, were much 14 longer in sense and shorter in antisense plants, and xylem tissue was 15 reduced in leaf and internode of sense plants. Antisense plants presented 16 a bushy phenotype, suggesting a possible effect of GAs on auxin 17 biosynthesis and/or transport. The main foliole of sense plants was longer, 18 although total leaf area was reduced. Leaf thickness was smaller in sense 19 and bigger in antisense plants due to changes in the spongy parenchyma. 20 Internode cell length was not altered in transgenic plants, indicating that in 21 citrus GAs regulate cell division rather than cell elongation. Interestingly, 22 the described phenotypes were not apparent when transgenic plants were 23 grafted on non-transgenic rootstock. This suggests that roots contribute to 24 the GA economy of aerial parts in citrus and opens the possibility of using 25 the antisense plants as dwarfing rootstocks.We thank J.A. Pina for technical assistance, and Dr. E. Carbonell and J. 34 Pérez for statistical analyses. This research was supported in part by grants CICYT AGL2003-01644, 1 INIA RTA04-13 and BIO2003-00151. C. 2 Fagoaga was recipient of an INIA-CCAA postdoctoral contract. I. Lliso was 3 recipient of an IVIA predoctoral fellowship. D.J. Iglesias and F.R. Tadeo 4 were recipients of INIA-CCAA and “Ramón y Cajal” MEC postdoctoral 5 contracts, respectively. 6 7Peer reviewe