Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin

Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin Sánchez-Pons et al. Sánchez-Pons et al. BMC Plant Biology 2011, 11:91 http://www.biomedcentral.com/1471-2229/11/91 (19 May 2011) Sánchez-Pons et al. BMC Plant Biology 2011, 11:91 http://www.biomedcentral.com/1471-2229/11/91 RESEARCH ARTICLE Open Access Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin Nuria Sánchez-Pons, Sami Irar, Nora García-Muniz and Carlos M Vicient* Abstract Background: Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death. Results: Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes. Conclusions: This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury. Background Maintenance of genome stability is of critical importance for all organisms. Genomic DNA is continuously subject to many types of damage resulting from endogenous factors (production of reactive oxygen species, stalled replication forks, etc.) or the action of exogenous agents (radiation, naturally occurring radioisotopes, chemical mutagens such as heavy metals, etc.) [1]. Doublestrand DNA breaks (DSBs) are one of the most serious forms of DNA damage, potentially causing chromosomal translocations and rearrangements [2]. In response to DSBs, cells initiate complex signalling pathways that activate DNA repair, cell-cycle arrest, and eventually cell death [3]. DSBs repair is mediated by two basic mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ) [4]. In HR, an intact copy of the damaged region (a sister chromatid, for example) acts as a template to repair the break. In NHEJ, DSBs are simply rejoined largely independently of the DNA * Correspondence: Department of Molecular Genetics, Centre for Research in Agricultural Genomics, Campus UAB, Edifici CRAG, Bellaterra (Cerdanyola del Vallés), 08193 Barcelona, Spain sequence. Bacteria and yeast usually employ HR whereas mammals and plants usually use NHEJ. In addition to the direct repair of DNA breaks, additional responses are activated during DNA-damage stress. For example, DNA damage in plant cells usually induces the accumulation of signal transduction intermediates such as nitric oxide, ROS or ethylene [5,6] and produces changes in the cytosolic-free Ca2+ [7]. It also induces cell cycle arrest, the inhibition of DNA and RNA synthesis, and a rapid protein turnover via the proteasome [8,9]. Additional reported effects are a reduction in the photosynthesis-related proteins [10], the accumulation of protective proteins such as pathogenesis-related protein-1 [11], the accumulation of protecting pigments [12], an increase in the expression of senescence- and cell death-associated genes [13] and the activation of different cellular detoxification mechanisms [14]. The regulation of all these responses is complex and involves different levels of regulation, including the modulation of transcriptional activity [15], post-transcriptional mechanisms (RNA processing, RNA silencing, etc.) [16-18] and post-translational modifications (protein phosphorylation, ubiquitination, SUMOylation, © 2011 Sánchez-Pons et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sánchez-Pons et al. BMC Plant Biology 2011, 11:91 http://www.biomedcentral.com/1471-2229/11/91 etc.) [19]. These processes are based on signal transduction initiated by sensor proteins that recognise the damage in the DNA and activate the transducers, which send the signal to the effector proteins [20]. The network of transcriptional, post-transcriptional and posttranslational modifications ensures temporally and spatially appropriate patterns of stress-responses. DNA topoisomerase I (TOPI) regulates the topological state of DNA by cleaving and re-joining one DNA strand and allowing DNA relaxation [21]. TOPI activity is essential in dividing cells to release the torsion created by the progression of DNA replication forks. The presence of active TOPI is essential for embryo development in Drosophila and mouse [22]. In plants, TOPI plays a similar basic role and, for example, the disruption of the two TOPI encoding genes in Arabidopsis thaliana is lethal [23]. Camptothecin (CPT) is a plant alkaloid that specifically binds TOPI, stabilising the complexes formed between DNA and TOPI [24]. The collisions between the trapped TOPI-CPT complexes and the replication fork during DNA replication produce DSBs which induce DNA damage responses [25]. In consequence, actively dividing cells are much more sensitive to CPT than non-dividing cells, a property that has been exploited in the treatment of cancer [24]. However, non-dividing cells are also sensitive to CPT as collisions of the RNA polymerase machinery with the TOPI-CPT complexes, although less frequent, are also able to produce DSBs [26]. CPT-mediated TOPI-DNA complexes can be degraded via the 26S proteasome pathway so, at low CPT concentrations, cells can survive [27]. However, in actively dividing cells the high number of collisions may exceed the capacity of the cells to eliminate TOPI-DNA complexes and the DNA repair capability of the cells and, under these circumstances, cell death is initiated. CPT has a similar effect on TOPI in plant and animals. For example, CPT inhibits, in vitro, the activity of TOPI extracted from maize immature embryos [28], produces the abortion of shoots and roots in Arabidopsis [23], and induces cell death in tomato cell cultures [29]. In this study, we profiled proteins and genes whose expression is changed in immature maize embryos as a consequence of the DNA damage produced by CPT. Immature embryos contain a high proportion of actively dividing cells and, in consequence, ar (...truncated)