1. Transcriptional analysis of phenanthrene treated plants revealed the induction of neoglucogenesis pathway associated with detoxification systems: possible utilisation in phytoremediation strategy.
Anne-Sophie Dumas1, Richard Berthomé2, Moez Shiri1, Jérôme Challis1 and Abdelhak El Amrani1
1CNRS, Université de Rennes 1, UMR 6553, Ecosystèmes-Biodiversité-Evolution, Rennes cedex, France,
2URGV, UMR INRA CNRS UEVE, 2, Rue Gaston Cremieux, CP5708, Evry cedex, France.
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern. In 2007, the European environmental agency estimated that 11% of the contaminated sites present a pollution by PAHs in Europe. To overcome the hazards associated with the PAHs, several conventional methods such as excavation, alteration or isolation could be used. These strategies are expensive and may impacts the environment. Phytoremediation is a cheaper emerging alternative that can be applied to soil decontamination by using the ability of plant to remove PAHs from soil and convert them into less toxic compounds. Indeed several plants (Frick, 1999) are known to tolerate high level of PAHs and to be implied in biodegradation processes. However, molecular and physiological events involved in PAH storage and degradation in plant are still poorly understood.
The aim of the present work is to decipher the early molecular processes involved in PAHs tolerance through a wide genome transcriptional analysis and to identify new factors that could contribute to improve phytoremediation.
Method
Transcriptome analysis was carried on using the CATMA V5 microarrays as described in Lurin et al Arabidopsis thaliana ecotype Columbia (Col 0) were grown in-vitro for 15 days on solid half-strength Murashige and Skoog (MS) medium, without any source of carbohydrates. Plantlets at stage 1.04 (Boyes, 2001) were then transferred on liquid half-strength MS medium containing 0.2mM of phenanthrene (prepared from a 700mM solution of phenanthrene in DMSO) or the same amount of DMSO. Plantlets were shaken over the experiment to ovoid oxygen limitation. Samples were harvested after 30 min, 2h, 4h and 24h of incubation. Figure 1 presents the experimental set up.
Biological pathways significantly over-represented in the list of differentially expressed genes were identified with the classification superviewer tool of the university of Toronto website ( using MAPMAN classification as a source.
Figure1: The blue spots represent the untreated samples (DMSO only) and the orange spots the treated samples (phenathrene+DMSO). The arrows represent the comparisons that are done; the down ones for the first biological repetition and the up ones for the second repetition.
Results
Figure 3: Histograms of significantly differentially expressed genes extracted from transcriptomic analysis of plants grown on phenanthrene supplemented medium compared to control. Up-regulated (red) and down-regulated (green) genes in phenanthrene treated plants are shown. Light shading indicates the proportion of genes similarly regulated in others points of the kinetic. Dark shading indicates the genes specifically regulated at indicated kinetic point. Numbers above and below each histogram correspond to the number of genes differentially up- or down- regulated at each kinetic point respectively.
Figure 2 shows representative impact of phenanthrene contaminated medium on plant development. Upon imbibitions, plants grown on phenanthrene supplemented medium showed a delayed growth, they displayed shorter roots and smaller leaves. Their leaves became pale green and, after 2 weeks, their growth stopped which lead to the plant death. However, when two weeks old plants were transferred on phenanthrene supplemented liquid medium, no macroscopic phenotype changes were observed after 24 hours of incubation  
Figure 2: 12 days old representative untreated plant (right) and phenanthrene (0.2mM) treated plant.
Figure 4: Biological pathways in which the up (fig 4.A) and down-regulated (fig 4.B) genes are significantly over-represented at each kinetic point. Group 1 (blue) represents the pathways in which genes are quickly up-regulated from 0h to 4h upon imbibitions of phenanthrene, group 2 (yellow) the pathways in which genes are up-regulated later between 4h and 24h and group 3 (green) the pathways corresponding to genes down-regulated at late stages, between 4 and 24h. The numbers correspond to frequency in our list of differentially genes normalized to frequency in Arabidopsis set.
Color legend: kinetic points A B
30min 2h 4h 8h
24h
Figure 5: Log base 2 ratios of 4 genes that could be involved in different phases of the green liver detoxification process (Fig 6.) at each kinetic point. These genes, belonging to the miscellaneous group (group 1, Fig 4A.) and the transport pathway for the last one (group 2, Fig 4A.), are quickly up-regulated upon phenanthrene imbibitions.
Phase I: transformation
Discussion
Despite no significant changes in phenotype were observed after 24 h of phenanthrene treatment, transcriptomic data obtained suggest for the first time that plant response to phenanthrene could be divided into steps: a fast and a delayed response, from 0 to 4h and 4h to 24h respectively. Interestingly, it is of high interest to notice that the number of the differentially expressed genes increased sharply between 4h and 8h indicating that a major changes are involved at this stage.
Through the analysis of transcriptomic data produced in our work, significant over-represented pathways fit well with the “green liver model” proposed by Sandermann in 1992, suggesting that this concept could be applied to the phenanthrene detoxification, at least during the early plant response, between 0 and 4h. Indeed, the gene encoding the cytochrome CYP705A5 involved in the transformation phase (phase I Fig.6) is highly up-regulated as early as 30 min. Interestingly, genes contributing to the conjugation phase (phase II Fig.6), such as genes encoding glycosyltransferase UGT76E11 and of the gluthatione-S-transferase GSTU4 are also quickly up-regulated, after 2h of imbibitions. In accordance with the green liver concept kinetics, one gene up-regulated 4h after phenanthrene treatment and encoding the ATP-binding cassette transporter ABCG31, belongs to the transport pathway. This last gene could be involved in the compartmentalization stage (Phase III, Fig.6). Moreover, between 0 to 4h, all the other pathways over-represented (group 1, Fig;4) are leading to glyoxylate cycle/gluconeogenesis responsible for the synthesis of soluble sugar. These sugars might not be synthesize only for the production of energy, but could be used as conjugates in the detoxification process (green liver phase I) and/or participate to a signaling cascade as it had been seen for several abiotic stresses (Rosa, 2009 ) .
We also suggest that the differentially regulated pathways involved in later stage (group 2 and 3, Fig.4) could be linked together. The TCA cycle and glucogenesis were up regulated at this later stage, which suggest that energy production (ATP) is induced and carbohydrates are realocated, probably to overcome stress induced by phenanthrene. In parallel, N-metabolism and amino-acids metabolism was down regulated. Indeed, the balance between carbon and nitrogen act as a signal in plants (Coruzzi, 2001). A lower level in carbon halts N-assimilation. This could explain why the N-metabolism pathway appeared differentially regulated. At this point, it is possible that the green liver process is not efficient enough so the plant has to remobilize the entire metabolism to respond to the phenanthrene induced stress.
Phase II: conjugation
Phase III: compartmentalization
Figure 6: Proposed model of “the green liver” process in plants .
Phase I-transformation: xenobiotics are chemically modified using oxidation, reduction, or hydrolysis to make these molecules more water soluble, this stage involved cytochrome P450 (CYP).
Phase II- conjugation: the xenobiotics are conjugates to endogenous molecules such as sugar. Glycosyltransferases (UGTs) transfer nucleotide-diphosphate-activated sugars like UDP-glucose to low-molecular-weight substrates. The conjugation step can also be performed by the gluthatione-S-transferases (GST) by attaching the tripeptide gluthatione to xenobiotics.
Phase III-compatmentalization: The conjugated xenobiotic is transfered to the vacuole by the ATP-binding cassette (ABC) transporters.
Conclusion
References
Frick et al. (1999), Assessment of Phytoremediation as an In-Situ Technique for Cleaning Oil-Contaminated Sites, pages 6-7
Lurin et al. (2004), Plant Cell, volume 16, pages
Boyes et al. (2001), Plant Cell, volume 13, pages
Sandermann et al. (1992), Trends in Biochemical Sciences, volume 17, pages 82-84
Rosa et al. (2009), Plant Signal Behavior, volume 4, pages 388–393.
Corruzi et al. (2001), Current Opinon. Plant Biology 4, 247–253.
During our experiment, the plant response to phenanthrene contamination could be divided into two stages:
an early stage, from 0 to 4h of treatment, in which the “green liver process” seems to be induced to initiate phenathrene detoxification;
a late stage, from 4h to 24h, in which the plant could not cope with contaminant anymore. In this stage, the stress response intensified.
Transcriptome data are currentlyvalidated by RT-qPCR. The involvement of genes encoding enzyme link to the green liver process will be explored through functional analysis. This work could lead us to find how to improve phenanthrene detoxification in plant and in-situ phytoremediation.
Aknowledgment
This work was supported by Bs Coatings (Aubevoye, France) and French Ministry of Higher Education and Research .