Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in Cichorium intybus L

<p>Abstract</p> <p>Background</p> <p>Somatic embryogenesis (SE) is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of <it>Cichorium intybus </it>L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E) plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE) under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory.</p> <p>Results</p> <p>Cytological analysis indicated that in K59 leaf explants the first cell divisions leading to SE were observed at day 4 of culture. In contrast, in C15 explants no cell divisions were observed and SE development seemed arrested before cell reactivation. Using mRNAs isolated from leaf explants from both genotypes after 4 days of culture under SE-inducing conditions, an E and a NE cDNA-library were generated by SSH. A total of 3,348 ESTs from both libraries turned out to represent a maximum of 2,077 genes. <it>In silico </it>subtraction analysis sorted only 33 genes as differentially expressed in the E or NE genotype, indicating that SSH had resulted in an effective normalisation. Real-time RT-PCR was used to verify the expression levels of 48 genes represented by ESTs from either library. The results showed preferential expression of genes related to protein synthesis and cell division in the E genotype, and related to defence in the NE genotype.</p> <p>Conclusion</p> <p>In accordance with the cytological observations, mRNA levels in explants from K59 and C15 collected at day 4 of SE culture reflected differential gene expression that presumably are related to processes accompanying early stages of direct SE. The E and NE library obtained thus represent important tools for subsequent detailed analysis of molecular mechanisms underlying this process in chicory, and its genetic control.</p

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.)

<p>Abstract</p> <p>Background</p> <p>In our laboratory we use cultured chicory (<it>Cichorium intybus</it>) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation <it>i.e</it>. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> showed that the use of the β-D-glucosyl Yariv reagent (β-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that β-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used β-GlcY to block SE in order to identify genes potentially involved in this process.</p> <p>Results</p> <p>Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. β-GlcY-treatment of explants blocked <it>in vitro </it>SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by β-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by β-GlcY-treatment: <it>AGP </it>(DT212818), <it>26 S proteasome AAA ATPase subunit 6 </it>(<it>RPT6</it>), <it>remorin </it>(<it>REM</it>), <it>metallothionein-1 </it>(<it>MT1</it>), two non-specific lipid transfer proteins genes (<it>SDI-9 and DEA1</it>), <it>3-hydroxy-3-methylglutaryl-CoA reductase </it>(<it>HMG-CoA reductase</it>), and <it>snakin 2 </it>(<it>SN2</it>). These results suggest that the 8 genes, including the previously-identified <it>AGP </it>gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory.</p> <p>Conclusion</p> <p>The use of two different chicory genotypes differing in their responsiveness to SE induction, together with β-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.</p

Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae)

<p>Abstract</p> <p>Background</p> <p>The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, <it>i.e</it>. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (<it>Cichorium intybus </it>L.) constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae.</p> <p>Findings</p> <p>Two bacterial artificial chromosome (BAC) libraries, CinS2S2 and CinS1S4, were constructed from <it>Hin</it>dIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and <it>S</it>₂<it>S</it>₂for the <it>S</it>-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and <it>S</it>₁<it>S</it>₄. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers.</p> <p>Conclusions</p> <p>This indicated that both BAC libraries are valuable tools for molecular studies in chicory, one goal being the positional cloning of the <it>S</it>-locus in this Asteraceae species.</p

Étude de l'autoincompatibilité chez la chicorée (Cichorium intybus L., Asteraceae) (cartographie génétique et physique du locus S)

L'autoincompatibilité (AI), généralement sous le contrôle d un seul locus, le locus S, est la stratégie la plus répandue pour empêcher l'autofécondation chez les plantes hermaphrodites. Les déterminants de la réaction d AI ont été identifiés dans quelques familles d Angiospermes. Dans celle des Asteraceae, ces déterminants sont encore inconnus. La chicorée (Cichorium intybus L., Asteraceae) présente un système d AI sporophytique. Afin d identifier les déterminants de l AI de cette espèce, une stratégie de clonage positionnel du locus S a été mise en place.Le locus S de la chicorée avait été cartographié à l'extrémité du groupe de liaison 2. Une carte haute densité a été obtenue à partir d une approche BSA assistée par marqueurs : 5 marqueurs AFLP ont été identifiés, dont un coségrégeant avec le locus S. A partir d une importante population (n = 2824), 2 cartes consensus haute résolution de la région du locus S ont été établies, l une pour les méioses femelles (intervalle de 0,6 cM) et l autre pour les méioses mâles (intervalle de 1,24 cM), en raison d un phénomène d hétérochiasmie.Deux banques BAC de chicorée ont été construites en collaboration avec le CNRGV. Elles ont permis d initier et d orienter la marche chromosomique vers le locus S. De plus, 6 clones BAC, soit 546 kb, ont été séquencés et analysés. Un rapport de 0,45 (méiose femelle) à 2 cM/Mb (méiose mâle) a été obtenu. La composition des séquences en éléments répétés et la fraction codante ont été étudiées (banques d EST de Cichorium sp. et de peptides d A.thaliana et de V.vinifera). Une relative conservation de la microsynténie a été observée entre ces gènes et leurs homologues chez 5 espèces Dicotylédones.Generally under the control of one locus, the S-locus, self-incompatibility (SI) is one of the most important strategies to prevent selfing in hermaphrodite plants. Determinants involved in the SI reaction have been identified in some Angiosperm families. In the Asteraceae family, determinants of SI are still unknown. Chicory (Cichorium intybus L., Asteraceae) is a sporophytic SI species. To identify determinants of chicory SI, a positional cloning strategy has been implemented.The chicory S-locus has been assigned to one end of linkage group 2. A high density map have been obtained from a marker assisted BSA (Bulk Segregant Analysis) approach: 5 AFLP markers were obtained, including one co-segregating with the S-locus. From a large population (n = 2.824), 2 consensus high-resolution map of the S-locus region were produced. Due to a heterochiasmy phenomenon, the S-locus was positioned in an interval of 0,6 cM or 1,24 cM considering female or male meiosis, respectively.Two BAC libraries of chicory were produced in collaboration with the CNRGV. They allowed us to start chromosome walking and to turn it toward the S-locus. Moreover, 6 BAC clones, corresponding to 546 kb, were sequenced and analyzed. A ratio of 0,5 Mb/cM for male meiosis and 2,2~Mb/cM for female meiosis were obtained. Repeated element composition and coding part of the sequences were studied (libraries of Cichorium sp. EST and Arabidopsis thaliana and Vitis vinifera peptides). A certain degree of conservation of microsynteny was observed between these genes and their homologues in five Dicotyledon species.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Effects of Nuclear Genomes on Anther Development in Cytoplasmic Male Sterile Chicories (Cichorium intybus L.): Morphological Analysis

The Cichorium intybus flower development in fertile, cytoplasmic male sterility (CMS 524) and various phenotypes carrying the 524 male sterile cytoplasm was investigated macroscopically and by light microscopy. The development was similar in fertile and in male sterile florets up to meiosis, and then it was affected in anther wall structure and pollen grain development in male sterile floret. In the male sterile plants, the tapetum intrusion after meiosis was less remarkable, the microspores started to abort at vacuolate stage, the connective tissue collapsed, and endothecium failed to expand normally and did not undergo cell wall lignification, which prevented anther opening since the septum and stomium were not disrupted. Crosses undertaken in order to introduce the CMS 524 into two different nuclear backgrounds gave rise to morphologically diversified progenies due to different nuclear-mitochondrial interactions. Macroscopic and cytological investigations showed that pollen-donor plants belonging to Jupiter population had potential capacity to restore fertility while the CC line could be considered as a sterility maintainer

Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in L-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in L"</p><p>http://www.biomedcentral.com/1471-2229/7/27</p><p>BMC Plant Biology 2007;7():27-27.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1913917.</p><p></p> c) and C15 (d, e, f); semi-thin (3 μm) sections from leaf explants at day 4 (a, b, d, e) and day 8 (c, f) of culture under SE-inducing conditions stained with toluidine blue. At day 4, reactivating (*) and reactivated (**) cells can be observed in K59 (a), a well as some recently divided cells (b; DC), whereas in C15 only some reactivating cells are present (c, d). At day 8, numerous somatic embryos (SE) can be observed in K59 explants. ST = stomate; VB = vascular bundle. Bars: 30 μm

A “Novel” Protocol for the Analysis of Hydroxycinnamic Acids in Leaf Tissue of Chicory (Cichorium intybus L., Asteraceae)

A “novel” protocol is presented for easy and reliable estimation of soluble hydroxycinnamate levels in Cichorium intybus L. leaf tissue in large-scale experiments. Samples were standardized by punching 6 discs per leaf, and hydroxycinnamates were extracted by submerging the discs in 80% ethanol with 5% acetic acid for at least 48 h in the darkness at 4°C. Residual dry mass of the discs was used for a posteriori correction of compound levels. Chlorophyll was eliminated by chloroform, and the aqueous phases were transferred to microplates, dried, and dissolved in 50% methanol for HPLC analysis and storage. An HPLC program of 8 min was developed for the analysis of the extracts. Comparisons with extractions of liquid nitrogen powders indicated that the novel extraction method was reliable. No degradation of the major hydroxycinnamates—caftaric, chlorogenic, and chicoric acids—was observed, during maceration at ambient temperatures, or after storage for 1 year

Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae)

Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis ofleaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology