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Differential expression of certain genes during somatic embryogenesis in leaf explants of Cichorium intybus

ABSTRACT: In Cichorium intybus, macroscopic and histological modifications were identified during in vitro cell reactivation and morphogenesis events. The relationships between these modifications and transcript profiles of genes were investigated. Two chicory genotypes derived from the Hungarian landrace Koospol, were tested: K59 is a responsive genotype (embryogenic), as it undergoes cell de- and re-differentiation that leads to somatic embryogenesis (SE). C15 is an unresponsive genotype (nonembryogenic), unable to express this morphogenetic pattern. Previously studies showed that addition of beta-D-glucosyl Yariv reagent (BGlcY), a synthetic phenyl-glycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in chicory root explants in a concentration-dependent manner with complete inhibition of induction occurring at 250 ÂµM of BGlcY (Chapmann et al. 2000a). A putative AGP (DT212818) encoding gene was previously found significantly over-expressed in the K59 as compared to the C15 and was suggested to be involved in chicory re-differentiation. To better understand the cell reactivation events, a treatment with BGlcY was applied in order to precipitate AGPs in muro and to block the in vitro cell re-differentiation processes. The two different genotypes together with BGlcY represented an original tool to discriminate cell reactivation events from cell fate determination. Microscopy, biochemical and transcriptome analyses showed that in vitro SE in chicory was blocked during BGlcY-treatment but cell reactivation still occurred. Consequently, AGP (DT212818) seems to be only important in cell fate determination leading to SE commitment. Gene expression profiles showed in addition to the AGP, other proteins could play a role in SE determination: 26S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein 1 (MT1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Cell wall events during BGlcY-treatment were observed and discussed. Gene expression of K59 explants induced (d4) was compared with gene expression of K59 explants without induction (d0). Gene expression of K59 explants induced in the presence of BGlcY (Y+; d4) was next compared with gene expression of K59 explants induced in the absence of BGlcY (Y-; d4). Similar comparisons were made for C15 leaf explants. To link up both genotypes, direct comparison was done between gene expression of K59 explants without induction (d0) and C15 explants without induction (d0). Three biological replicates were used for each analysed condition. Dye labelling for each paired sample was reversed in two subsequent individual hybridizations giving rise to a total of six hybridizations per condition except for the K59_D0_vs_C15_D0 condition.

ORGANISM(S): Cichorium intybus

SUBMITTER: Ludovic Huot

PROVIDER: E-GEOD-15502 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion paper, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or non-genotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and non-genotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4h and collected 0h, 4h and 20h post-exposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24h. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as non-genotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24h, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. This experiment examined the whole genome transcriptional response of TK6 cells exposed to DMSO (+S9) and 10 μg/ml BaP (-S9) (both compared to DMSO (-S9)) for 4 hours followed by a 20h in fresh media (24h time point), in addition to 7.5 mM dexamethasone (+S9) (compared to DMSO (+S9)). Each condition had 3 biological replicates, which were pooled for gene expression analysis. There were a total 3 samples (6 arrays) included in the final analysis using a two-colour dye swap design.

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Differential expression of certain genes during somatic embryogenesis in leaf explants of Cichorium intybus

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