Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin
© Sánchez-Pons et al; licensee BioMed Central Ltd. 2011
Received: 29 October 2010
Accepted: 19 May 2011
Published: 19 May 2011
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.
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.
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.
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.) . Double-strand DNA breaks (DSBs) are one of the most serious forms of DNA damage, potentially causing chromosomal translocations and rearrangements . In response to DSBs, cells initiate complex signalling pathways that activate DNA repair, cell-cycle arrest, and eventually cell death . DSBs repair is mediated by two basic mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ) . 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 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+ . 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 , the accumulation of protective proteins such as pathogenesis-related protein-1 , the accumulation of protecting pigments , an increase in the expression of senescence- and cell death-associated genes  and the activation of different cellular detoxification mechanisms . The regulation of all these responses is complex and involves different levels of regulation, including the modulation of transcriptional activity , post-transcriptional mechanisms (RNA processing, RNA silencing, etc.) [16–18] and post-translational modifications (protein phosphorylation, ubiquitination, SUMOylation, etc.) . 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 . The network of transcriptional, post-transcriptional and post-translational 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 . 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 . In plants, TOPI plays a similar basic role and, for example, the disruption of the two TOPI encoding genes in Arabidopsis thaliana is lethal . Camptothecin (CPT) is a plant alkaloid that specifically binds TOPI, stabilising the complexes formed between DNA and TOPI . The collisions between the trapped TOPI-CPT complexes and the replication fork during DNA replication produce DSBs which induce DNA damage responses . 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 . 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 . CPT-mediated TOPI-DNA complexes can be degraded via the 26S proteasome pathway so, at low CPT concentrations, cells can survive . 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 , produces the abortion of shoots and roots in Arabidopsis , and induces cell death in tomato cell cultures .
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, are particularly sensitive to CPT. The combination of microarray and two-dimensional gel electrophoresis protein analysis allowed us to identify molecular events that are regulated during DNA repair responses in plants at different levels: transcriptional, post-transcriptional, translational and post-translational. We identified candidate genes and proteins which may be specifically involved in the DNA repair responses.
Camptothecin induces DNA damage responses in maize immature embryos but not an extensive cell death process
Maize caryopses were collected 15 days after pollination and their dissected embryos incubated, in the dark, in culture medium with or without 50 μM camptothecin (CPT). After 8 days of culture, the germination rates of treated and untreated embryos were not significantly different (24% ± 5 in control and 20% ± 6 in treated embryos) and their morphological characteristics were similar.
Nucleases are involved in DNA damage responses  and in cell death . In plants, cell death-related nucleases have been classified according to their cationic cofactors, as Ca2+ or Zn2+-dependent . The ability of CPT to induce nuclease activities in maize embryos was tested using in-gel nuclease activity assays (Figure 1C and 1D). An increase in the activity of a Ca2+-dependent nuclease of about 32 kDa was clearly evident after 3 days of CPT treatment using an assay buffer containing 1 mM CaCl2, being only slightly reduced after 8 days of treatment (Figure 1C), and was higher in the embryo axis compared to scutellum (Figure 1D). In contrast, no zinc-dependent nuclease activity was detected using 1, 2 or 5 mM Zn2+ (results not shown).
The CPT-induced Ca2+-dependent nuclease could be involved in DNA repair but also in programmed cell death (PCD). PCD is usually characterised by inter-nucleosomal genomic DNA fragmentation, producing, after gel electrophoresis, a characteristic DNA ladder pattern . The results of electrophoresis of genomic DNA extracted from treated embryos was not significantly different to that observed with untreated embryos, showing a certain DNA ladder (Figure 1E). The same analyses using DNA extracted separately from embryo axis and scutellum clearly show that the DNA ladder was only present in the scutellum sample (Figure 1F). Degradation in genomic DNA extracted from scutellum has been previously observed in maize . Cells in the scutellum close to the embryo axis undergo PCD as a normal part of seed development and this may explain the observed DNA ladder . Exposure to 50 μM CPT did not, however, produce a significant change in the DNA integrity in the embryo axis or scutellum. This suggests that the CPT-induced Ca2+-dependent nuclease is involved in DNA repair and not in cell death.
These results indicate that, under the conditions used here, CPT induced DNA repair responses in maize embryos but not an extensive cell death process.
Transcriptional responses to CPT-induced DNA damage
Genes up-regulated by CPT-induced DNA damage.
Probe Set ID
Arab.ortholog gene AGI code (BLAST core)
Cell growth & division
Mob1-like protein/cell cycle checkpoint regulation
Putative hydroxyproline-rich glycoprotein
Vegetative cell wall protein gp1 precursor
Defense and stress responses
Class III peroxidase precursor
Acidic classI chitinase
Glutathione S-transferase GST 41
Glutathione S-transferase GST 36
DNA replication, recombination and repair
Ribonucleoside-diphosphate reductase small chain
Ribonucleotide reductase R1 (large subunit)
Putative DNA repair protein rhp16
Replication protein A2
Acetyltransferase, GNAT family protein
Putative X-ray induced gene 1 (XRI-1)
NADH dehydrogenase I subunit N
SC3 protein/Secretory carrier-associated membrane protein
Plastid ADP-glucose pyro-phosphorylase large subunit
Purple acid phosphatase 1
Putative Tat binding prot.1 (TBP-1)-interact.prot.(TBPIP)
PI31 Proteasome inhibitor-like protein
Signal transduction andgene expression
NAC domain-containing protein 77
NAC domain-containing protein 21/22
Putative Rop family GTPase, ROP7 (AtROP9)
Transposon protein Pong subclass
Leucine-rich repeat, cysteine-containing protein
Histidine kinase-like ATPase superfamily
Genes down-regulated by CPT-induced DNA damage.
Probe Set ID
Arab. ortholog gene AGI code (BLAST score)
Cell growth and division
SMC-like domain containing protein
Frizzy-like protein/WD-repeat cell cycle regulatory protein
Cyclin IIZm (CYCA1;1)
Cyclin IIIZm (CYCB2)
Syntaxin-related protein KNOLLE
Cyclin IaZm (cyclin-B1;2/CYC1BAT)
Patellin-5/SEC14 cytosolic factor-like
Microtubule-associated protein RP/EB family member 3
Defense and stress responses
Dehydration-responsive protein RD22
Vacuolar protein sorting-associated protein
Vacuolar protein sorting 13C protein-like
Endoglucanase 1 precursor
Signal transduction and gene expression
RRM-containing protein SEB-4
Myb-like DNA-binding domain containing protein
Serine/arginine repetitive matrix protein 1
Transcriptional regulatory protein algP
Homeobox transcription factor KNOTTED1
VEF family protein/embryonic flower 2
Rough sheath1 (RS1)/Homeo-box protein knotted-1-like
Embryogenic callus protein 98b/HMG1/2-family protein
Growth-regulating factor 8-like (atGRF2)
B3 domain containing DNA binding protein
Putative receptor protein kinase (ERECTA)
ATROPGEF7 Rho guanyl-nucleotide exchange factor
Protein binding protein/ankyrin repeat family protein/hox1a
Zinc finger (C3HC4-type RING finger)-like protein
Lipid binding protein
Histidine kinase-like ATPases superfamily
Alpha-amylase inhibitor, lipid transfer & seed storage protein
Uncharacterized plant-specific domain TIGR01568 protein
Putative mitochondrial glycoprotein
Glycin-rich protein 3 (ZmGrp3)
Unknown protein/Armadillo-type fold
Glycine rich protein 3
CPT treatment induced the expression of genes involved in DNA repair and DNA damage responses as, for example:
- Two subunits of the ribonucleotide reductase: involved in the DNA repair processes .
- RAD51: encodes a protein required for meiosis and HR repair . Maize mutants in two RAD51 maize genes are hypersensitive to radiation . The Arabidopsis gene AtRAD51a is transcriptionally up-regulated by DSB-inducing agents and seems to be required for HR repair after bleomycin treatment .
- Rpa2: encodes a protein that is part of a heterotrimeric protein complex that specifically binds single-stranded DNA (ssDNA) and plays multiple roles in DNA metabolism, including DNA repair and recombination . RPA genes are transcriptionally induced in Aspergillus nidulans exposed to CPT .
- TBPIP1: encodes a protein involved in chromosome pairing and segregation . In humans, TBPIP1 enhances the strand exchange mediated by RAD51 . In Arabidopsis, the TBPIP1 gene is transcriptionally induced by DNA damage .
- XRI-1: encodes a protein essential for meiosis and that plays a role during HR in Arabidopsis . This gene is highly and rapidly transcriptionally induced by X-ray radiation and is also highly induced by other DSBs-inducer agents . The encoded protein is probably part of the meiotic recombination complex MND1/AHP2, which collaborates with RAD51 in the DNA strand invasion during recombination .
- Acetyltransferase, GNAT family protein: some yeast GNAT family members are involved in DSBs repair .
- Rph16: encodes a protein similar to RAD16 and is involved in the nucleotide excision repair of UV damage .
CPT treatment repressed the expression of genes involved in cell cycle, cell division and cell growth (Table 2). For example:
- Three cyclins: IaZm, IIZm and IIIZm.
- Shugosin-1: encodes a protein involved in the maintenance of centromeric cohesion of sister chromatids during meiosis and mitosis. Depletion of the human Sgo1 gene produces mitotic cell cycle arrest .
- Patellin-5: patellins are involved in vesicle trafficking events. The Arabidopsis patellin PATL1 has been associated with the formation of the cell-plate during cytokinesis .
- Knotted1 (Kn1): encodes a homeo-domain protein involved in the regulation of leaf cell development .
- Microtubule-associated protein RP/EB family member 3: encodes a protein that binds to the end of the microtubules and is important in maintaining the structure of the mitotic spindle .
- Growth regulating factor 8-like: encodes a protein involved in leaf and cotyledon growth in Arabidopsis .
- Rough sheath1: encodes a protein involved in cell differentiation .
- Frizzy-like protein/WD-repeat cell cycle regulatory protein: encodes a protein similar to the tomato CCS52B that probably is involved in cell-cycle control during mitosis .
Alterations in maize embryo proteome in response to CPT
List of the spots/proteins identified by mass spectrometry.
Mw (kD) (Exp/Theo)
Chloroplast Cu/Zn superoxide dismutase (maize)
Cytosolic ascorbate peroxidase (maize)
Glutathione S-transferase 19 (maize)
Pathogenesis-related protein 1 (maize)
Bet v I allergen (maize)
26S proteasome non-ATPase regulatory subunit 4 (maize)
26S proteasome non-ATPase regulatory subunit 8 (maize)
Eukaryotic translation initiation factor eIF-5A (maize)
Eukaryotic translation initiation factor eIF-6 (maize)
DNA replication, recombination & repair
Histone H2B (maize)
DEAD-family RNA helicase (rice)
Glycine-rich RNA-binding protein 8 (maize)
RNA recognition motif containing protein (maize)
Cytosolic GAPDH (maize)
Glyceraldehyde-3-phosphate dehydrogenase (Arabidopsis)
Glyceraldehyde-3-phosphate dehydrogenase (Arabidopsis)
Triosephosphate isomerase 1 (maize)
Protein-G β-subunit (rice)
Syntaxin 6 (rice)
Mitochondrial ATP synthase precursor (maize)
Embryo and seed storage
globulin 2 (maize)
globulin 2 (maize)
globulin 2 (maize)
cupin domain containing protein (rice)
r40c1 protein (rice)
Some of the identified proteins are involved in antioxidant responses. Antioxidant activity protects against ROS accumulation, which can be produced by a variety of stresses, including DNA damage . In mammals, CPT induces the accumulation of antioxidant enzymes in the nucleus . The accumulation of two proteins involved in pathogenesis responses, PR1 and Betv1, was observed in response to CPT. They are also induced by abiotic stresses such as heavy metals  and UV radiation .
The accumulation of at least two 26s proteasome regulatory subunits is altered in response to CPT, one increased and the other reduced. Interestingly, CPT-TOPI-DNA complexes may be degraded by the ubiquitin-dependent pathway .
We observed changes in the accumulation of several proteins involved in RNA metabolism or RNA binding proteins:
- RraA: an RNaseE inhibitor which may be involved in the degradosome complex in E. coli [69, 70]. Regulation of RraA by DNA damage stress could be related to changes in the regulation of RNA homeostasis.
- Glycine-rich RNA-binding protein 8: some glycine-rich RBPs in Arabidopsis (GR-RBPs) are significantly induced by cold, drought and salinity, whereas others are repressed by other sources of stresses .
- RNA recognition motif-containing (RRM) protein: RRM-containing proteins are involved in most post-transcriptional gene expression processes (i.e. mRNA and rRNA processing, RNA export and stability) .
We also observed an increase in the accumulation of two spots corresponding to eukaryotic elongation factors. Experiments in yeast and mammals demonstrate that translation initiation factor 5A (eIF5A) is actually involved in mRNA nucleus-cytoplasm export and not translation, specifically regulating genes involved in cell growth and proliferation, and in cell death . In Arabidopsis, AteIF5A/AtFBR12 (At1g26630) promotes PCD associated with the hypersensitive pathogen response , and AteIEF5A-1 (At1g13950) has been associated with PCD during xylogenesis . Thus, regulation of eiF5A by CPT suggests it is involved in cell cycle and PCD regulation.
Lack of correlation between CPT-induced changes in protein abundance and changes in mRNA accumulation
GeneChip array data analysis of the maize genes encoding the proteins identified by mass spectrometry.
Affymetrix chip data
Probe Set ID
Chloroplast Cu/Zn superoxide dismutase
Cytosolic ascorbate peroxidase 1
Glutathione S-transferase GST19
26S proteasome non-ATPase regulatory subunit 4
26S proteasome non-ATPase regulatory subunit 8
Eukaryotic translation initiation factor eIF-5A
Eukaryotic translation initiation factor eIF-6
Triosaphosphate isomerase 1
Regulator of RNAse activity A
Regulator of RNAse activity A
DEAD-family RNA helicase
Glycine-rich RNA-binding protein 8
RNA recognition motif containing protein
Pathogenesis-related protein 1
Bet v I allergen
Mitochondrial ATP synthase precursor
Cupin domain containing protein
Our aim was to identify new elements involved in cellular responses to genomic damage in plants, using CPT as a toxic agent and applying transcriptomic and proteomic approaches to identify the genes, proteins and cellular mechanisms involved. We identified a series of genes and proteins whose expression/accumulation significantly change in response to CPT, although the identified genes do not correspond to the identified proteins. These differences may be a consequence of the different sensitivity of the methods. Moreover, the level of protein accumulation does not necessarily agree with the level of mRNA expression. This incongruent expression between mRNAs and proteins has been observed by other groups, in other species and experimental conditions [78–80] and is most likely a result of the biology of gene expression which includes various levels of regulation during protein synthesis: post-transcriptional, translational, and post-translational. Thus, integrated analysis of both mRNAs and proteins is crucial to gain further insights into complex biological systems.
The basic mechanism of action for CPT has been well-studied and characterised in animal cells . CPT generates replication-mediated DSBs in DNA which in turn induce DNA repair, cell cycle arrest and, under certain circumstances, cell death. Under our conditions, CPT did not induce extensive cell death in maize embryos, as demonstrated by TUNEL staining which only appeared in some cells in the embryo axis after CPT-treatment. At the developmental stage analyzed here, cells in the scutellum divide at a very limited rate, but cells in the embryo axis divide rapidly. This difference may explain the higher sensitivity to CPT of the cells in the embryo axis.
Two basic mechanisms of DSBs DNA repair have been described: homologous recombination and non-homologous end joining . Our transcriptomic analysis identified the induction of some genes already known to be involved in DNA repair. Interestingly, most of them are involved in the HR repair pathway, suggesting that this is the main mechanism for DSBs repair in maize embryos, at least in response to CPT. CPT also produces an increase of a 32 kDa calcium-dependent nuclease activity. However, this nuclease is unlikely to be involved in the extensive fragmentation of the genomic DNA observed in different cell death processes as extensive DNA fragmentation was not observed. Nucleases are also involved in most DNA repair mechanisms, including HR . These data suggests that the 32 kDa nuclease activity observed may be involved in the DNA repair process.
CPT induces reversible or permanent cell-cycle arrest in G2-M phase in human and other cells  and produces major alterations in the expression of cell-cycle regulatory genes . We found that CPT reduces the expression of several mitosis-related genes. In addition, we observed a reduction in the accumulation of the histone H2B involved in the structure of chromatin, and changes in the accumulation of two eukaryotic translation initiation factors which seem to also be involved in the cell-cycle process . These results suggest that, in maize embryos, one of the cellular responses to CPT is the arrest of cell division.
In addition to more specific processes, DNA damage induces general stress mechanisms in maize embryos. For example, we observed changes in the expression and accumulation of proteins involved in ROS processing (glutathione S-transferase, Class III peroxidase precursor, chloroplast Cu/Zn superoxide dismutase, cytosolic ascorbate peroxidase), enzymes involved in glycolic metabolism (glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase 1) and in pathogen responses (pathogenesis-related protein1 and Bet v I allergen). Pathogen resistance is increased after DNA damage induction, indicating a cross-link in DNA damage (and maybe other abiotic stresses) and defense responses .
An increasing number of studies combining proteomics and transcriptomics clearly demonstrate that mRNA and protein accumulation are not always correlated [86–88]. For instance, in yeast 73% of the variance in protein abundance is explained by the translation mechanism and only 27% due to variations in mRNA concentration . Protein abundance is influenced by several factors at the post-transcriptional, translational, and post-translational levels. For example, there is a time lag between transcription and translation in which introns are excised and the transcripts are moved from the nucleus to the cytoplasm, and translation rates may be influenced by ribosome, tRNA and amino acids availability, codon usage or accessory protein binding association . In addition, protein abundance is also influenced by post-translational processes such as glycosylation, phosphorylation and proteolytic processing.
Our proteome analysis indicated differences in the abundance (up and down) of the encoded proteins of 24 genes whose mRNA levels do not significantly change in response to CPT (Table 4). It is possible that CPT induces the transcription of some genes only during the first hours of treatment, and after three days of treatment the mRNA levels are similar to the control but the abundance of the encoded protein is higher. Differences in the translation rate may also explain the lack of correlation. In animals, post-transcriptional regulation of gene expression during the stress response means specific stress-induced transcripts receive the highest transcriptional priority . Interestingly, some of the spots identified in the proteomic analysis correspond to proteins associated with RNA metabolism and RNA binding proteins, and may be involved in the regulation of mRNA translation.
Many post-translational processes affect the position of a protein in 2D gels such that the protein appears as differentially accumulated in a proteomic analysis. We have identified changes in genes and proteins involved in protein modification and post-translational regulation. For example, the accumulation of at least two 26s proteasome regulatory subunits is altered in response to CPT and the expression of the proteasome inhibitor-like protein PI31 is increased. Ubiquitin/proteasome-mediated protein degradation plays a central role in the regulation of several aspects of plant development and stress responses  and our data indicate that it may also be involved in regulating DNA damage responses. In fact, there are evidences that CPT-TOPI-DNA complexes may be degraded by the ubiquitin-dependent pathway in mammals and yeast [27, 93]. Our data suggest that a similar situation may occur in plants. Moreover, the expression of embryonic flower 2 is repressed, a gene encoding a protein homologous to Drosophila Polycomb genes which mediate the epigenetic control of homeotic gene expression .
The role of several of the genes identified in the transcriptomic analysis is unknown. These genes may play a role in DNA damage detection and repair mechanisms, especially those genes that are only induced in response to genomic damage and not in response to other types of stress. Unfortunately the data currently available in maize does not allow us to determine which of them are specifically induced by DNA damage, but many of the maize identified genes have clear homologues in Arabidopsis (Table 1 and 2). Microarray analyses in Arabidopsis have been used to study the effects of several abiotic stresses, including two DNA damage agents, bleomycin  and gamma radiation . Examining microarray databases  we identified eight Arabidopsis genes homologous to maize CPT-induced genes and exclusively induced by DNA damage: At5g02220, of unknown function; At1g13330, encoding TBP-1 tat binding protein; At5g48720, encoding an X-ray induced gene required for post-meiotic stages of pollen development and for male and female meiosis; At3g27060 and At2g21790, encoding the ribonucleotide reductase (RNR) small and large subunit, respectively; At5g20850, encoding AtRAD51; and two genes, At5g18270 and At3g04060, encoding NAC transcriptions factors. NAC proteins constitute one of the largest families of plant-specific transcription factors, and the family is present in a wide range of land plants . These two NAC proteins are interesting candidates for a regulatory role in DNA damage responses in plants.
The integration of microarray and proteomic analyses provides new data on DNA damage responses in plants. This is a complex process involving DNA repair and arrest of cell-cycle, but also general stress responses. Post-translational processing and the regulation of mRNA translation seem to have an important role in DNA damage responses.
Plant material and treatments
Maize (Zea mays L cv W64A pure inbred line) was grown under controlled conditions (16 h light, 28°C). Immature embryos (15 days after pollination) were extracted in sterile conditions and placed on MS plates (4.4% (w/v) Murashige and Skoog medium, 0.8% (w/v) Gelrite) supplemented (or not) with 50 μM camptothecin (Sigma-Aldrich) and maintained in a growth chamber at 26°C in darkness.
Embryos were collected, fixed in ethanol-formaldehyde-acetic acid (80:3.5:5) for 1 h at room temperature, followed by 1 week at 4°C, and then stored in 70% ethanol at 4°C. Fixed samples were embedded in paraplast, de-waxed with Histo-Clear II (National Diagnostic, UK), re-hydrated in an ethanol series and equilibrated in 0.02 M citric acid-0.16 M Na2HPO4, pH 7.0. TUNEL assays were done using the In Situ Cell Death Detection kit (Roche) according to the supplier's protocol for difficult tissues. In negative controls, the TdT enzyme was omitted, and the positive controls were treated with DNase I for 10 min. Experiments were repeated three times.
RNA extraction and quantification
Total RNA was isolated from frozen samples using the lithium chloride method. DNase digestion of contaminating DNA in the RNA samples was done using RNase-Free DNaseI. Final RNA purification was performed using the RNeasy Mini Kit (Qiagen) according to standard protocols. RNA was quantified with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies). RNA quality was assessed with a 2100 Bioanalyzer from Agilent Technologies.
Affymetrix GeneChip hybridization/Microarray analysis
Gene expression was analyzed using the Affymetrix GeneChip® Maize Genome Array, which contains probe sets to interrogate 13,339 genes, performing four independent biological replicates. cDNA synthesis, probe labeling, array hybridization and data analysis were as described by Bannenberg and col. , in the Genomics Service of the Centro Nacional de Biotecnologia (CNB-CSIC, Madrid). Raw data and normalised data were deposited at the ArrayExpress data library (http://www.ebi.ac.uk/arrayexpress/) under accession number E-MEXP-2702. Differential expression was considered following the p < 0.05 and 2.0 fold change as the criteria of significance. Functional categories of the genes were determined based on Gene Ontology data. We used the Fisher's Exact Test (p ≤ 0.05) and ANOVA (p ≤ 0.01) to determine the significant differences in the functional categories among up- and down-regulated genes.
Real time quantitative RT-PCR
Sequences of primers used in the real-time PCR experiments.
In-gel Nuclease Activity Assay
Immature maize embryos were ground in liquid nitrogen and resuspended in extraction buffer (150 mM Tris-HCl, pH 6.8, 0.5 mM PMSF, 20 μM leupeptin). The homogenate was clarified by centrifugation at 12,000 g for 5 min at 4°C. In-gel nuclease activity was measured according to Thelen and Northcote  using 10 μg of protein in 12.5% SDS-PAGE containing 50 μg/mL of single-stranded calf thymus DNA (Sigma) and 50 μg/mL bovine fibrinogen (Sigma). After electrophoresis, gels were washed twice in 25% (v/v) isopropanol, 10 mM Tris-HCl pH 7.0 for 30 min and twice in 10 mM Tris-HCl, pH 7.5 for 30 min. Gels were incubated overnight at 37°C with gentle agitation in 10 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 1 mM MgCl2 for Ca2+/Mg2+-dependent activity or in 25 mM NaAc/HAc pH 5.5, 1, 2 or 5 mM ZnSO4, for Zn2+-dependent activity. Nuclease activity was detected by staining the gel with 1 μg/mL (w/v) ethidium bromide for 15 min and observed under UV.
Two-dimensional gel electrophoresis
Maize embryos were ground in liquid nitrogen and crude protein extracts were solubilised in 1.2 ml buffer 1 (7 M urea, 2 M thiourea, 4% CHAPS, 4% Triton X-100, 18 mM Tris-HCl pH 8) in the presence of 53 u/ml DNase I, 4.9 u/ml RNaseA and a cocktail of protease inhibitors (1 mM PMSF, 50 μM leupeptin, 1 μM pepstatin, 10 μM E-64, 10 μg/ml aprotinin). After 20 min incubation at 4°C, DTT at a final concentration of 14 mM was added and samples were centrifuged 10 min at 35000 g at 4°C. 2-DE analysis was performed basically as previously described  using pH 3-11, 24 cm immobilised pH gradient (IPG) strips (Immobiline DryStrips, GE Healthcare) for the first dimension. The optimal parameters for spot detection were: smooth = 4, saliency = 1.0 and minimum area = 5. To evaluate protein expression differences among gels, relative spot volume (% Vol.) was used. Protein abundance variation was validated by Student's t-Test (p < 0.05).
In-gel digestion of proteins and MS and MS/MS spectra
Proteins were in-gel digested with trypsin and tryptic peptides were extracted and analyzed by MALDI-TOF/MS (4700 Proteomics Analyzer, Applied Biosystems) or LC-ESI-QTOF (Q-TOF Global, Micromass-Waters) mass spectrometers in the Proteomics Platform (PCB) of the University of Barcelona as previously described .
List of Abbreviations
DNA topoisomerase I
double strand breaks
programmed cell death
in situ detection of fragmented DNA
tandem mass spectrometry
false discovery rate
This work was supported by grants BIO2004-01577 and AGL2009-09151 from Plan Nacional de Investigación Científica y Técnica, by the Centre CONSOLIDER on Agrigenomics and by the Xarxa de Referencia en Biotecnologia of the Generalitat de Catalunya. NSP was supported by a grant from the CSIC.
- Britt AB: DNA Damage and Repair in Plants. Annu Rev Plant Physiol Plant Mol Biol. 1996, 47: 75-100. 10.1146/annurev.arplant.47.1.75.PubMedView ArticleGoogle Scholar
- Rich T, Allen RL, Wyllie AH: Defying death after DNA damage. Nature. 2000, 407: 777-783. 10.1038/35037717.PubMedView ArticleGoogle Scholar
- Jackson SP: Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002, 23: 687-696. 10.1093/carcin/23.5.687.PubMedView ArticleGoogle Scholar
- West CE, Waterworth WM, Sunderland PA, Bray CM: Arabidopsis DNA double-strand break repair pathways. Biochem Soc Trans. 2004, 32: 964-966. 10.1042/BST0320964.PubMedView ArticleGoogle Scholar
- A.-H.-Mackerness S, John CF, Jordan B, Thomas B: Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide. FEBS letters. 2001, 489: 237-242. 10.1016/S0014-5793(01)02103-2.PubMedView ArticleGoogle Scholar
- A.-H.-Mackerness S, Surplus SL, Blake P, John CF, Buchanan-Wollaston V, Jordan BR, Thomas B: Ultraviolet-B-induced stress and changes in gene expression in Arabidopsis thaliana: role of signalling pathways controlled by jasmonic acid, ethylene and reactive oxygen species. Plant, Cell & Environment. 1999, 22: 1413-1423. 10.1046/j.1365-3040.1999.00499.x.View ArticleGoogle Scholar
- Frohnmeyer H, Loyall L, Blatt MR, Grabov A: Millisecond UV-B irradiation evokes prolonged elevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures. The Plant Journal. 1999, 20: 109-117. 10.1046/j.1365-313X.1999.00584.x.PubMedView ArticleGoogle Scholar
- Logemann E, Wu SC, Schroder J, Schmelzer E, Somssich IE, Hahlbrock K: Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes. Plant J. 1995, 8: 865-876. 10.1046/j.1365-313X.1995.8060865.x.PubMedView ArticleGoogle Scholar
- Molinier J, Oakeley EJ, Niederhauser O, Kovalchuk I, Hohn B: Dynamic response of plant genome to ultraviolet radiation and other genotoxic stresses. Mutat Res. 2005, 571: 235-247.PubMedView ArticleGoogle Scholar
- Desimone M, Wagner E, Johanningmeier U: Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis. Planta. 1998, 205: 459-466. 10.1007/s004250050344.View ArticleGoogle Scholar
- Surplus SL, Jordan BR, Murphy AM, Carr JP, Thomas B, Mackerness SAH: Ultraviolet-B-induced responses in Arabidopsis thaliana: role of salicylic acid and reactive oxygen species in the regulation of transcripts encoding photosynthetic and acidic pathogenesis-related proteins. Plant, Cell & Environment. 1998, 21: 685-694. 10.1046/j.1365-3040.1998.00325.x.View ArticleGoogle Scholar
- Kalbina I, Strid A: Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes. Plant, Cell & Environment. 2006, 29: 754-763. 10.1111/j.1365-3040.2005.01436.x.View ArticleGoogle Scholar
- John CF, Morris K, Jordan BR, Thomas B, A-H-Mackerness S: Ultraviolet-B exposure leads to up-regulation of senescence-associated genes in Arabidopsis thaliana. Journal of Experimental Botany. 2001, 52: 1367-1373. 10.1093/jexbot/52.359.1367.PubMedView ArticleGoogle Scholar
- Casati P, Walbot V: Rapid transcriptome responses of maize (Zea mays) to UV-B in irradiated and shielded tissues. Genome Biol. 2004, 5: R16-10.1186/gb-2004-5-3-r16.PubMedPubMed CentralView ArticleGoogle Scholar
- Ricaud L, Proux C, Renou JP, Pichon O, Fochesato S, Ortet P, Montane MH: ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis. PLoS One. 2007, 2: e430-10.1371/journal.pone.0000430.PubMedPubMed CentralView ArticleGoogle Scholar
- Anderson P, Kedersha N: Stressful initiations. J Cell Sci. 2002, 115: 3227-3234.PubMedGoogle Scholar
- Mazzucotelli E, Mastrangelo AM, Crosatti C, Guerra D, Stanca AM, Cattivelli L: Abiotic stress response in plants: When post-transcriptional and post-translational regulations control transcription. Plant Science. 2008, 174: 420-431. 10.1016/j.plantsci.2008.02.005.View ArticleGoogle Scholar
- Pothof J, Verkaik NS, van IW, Wiemer EA, Ta VT, van der Horst GT, Jaspers NG, van Gent DC, Hoeijmakers JH, Persengiev SP: MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. Embo J. 2009, 28: 2090-2099. 10.1038/emboj.2009.156.PubMedPubMed CentralView ArticleGoogle Scholar
- Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ: ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science. 2007, 316: 1160-1166. 10.1126/science.1140321.PubMedView ArticleGoogle Scholar
- Cimprich KA, Cortez D: ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol. 2008, 9: 616-627. 10.1038/nrm2450.PubMedPubMed CentralView ArticleGoogle Scholar
- Champoux JJ: DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem. 2001, 70: 369-413. 10.1146/annurev.biochem.70.1.369.PubMedView ArticleGoogle Scholar
- Lee MP, Brown SD, Chen A, Hsieh TS: DNA topoisomerase I is essential in Drosophila melanogaster. Proc Natl Acad Sci USA. 1993, 90: 6656-6660. 10.1073/pnas.90.14.6656.PubMedPubMed CentralView ArticleGoogle Scholar
- Takahashi T, Matsuhara S, Abe M, Komeda Y: Disruption of a DNA topoisomerase I gene affects morphogenesis in Arabidopsis. Plant Cell. 2002, 14: 2085-2093. 10.1105/tpc.001925.PubMedPubMed CentralView ArticleGoogle Scholar
- Pommier Y: Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer. 2006, 6: 789-802. 10.1038/nrc1977.PubMedView ArticleGoogle Scholar
- Piret B, Piette J: Topoisomerase poisons activate the transcription factor NF-kappaB in ACH-2 and CEM cells. Nucleic Acids Res. 1996, 24: 4242-4248. 10.1093/nar/24.21.4242.PubMedPubMed CentralView ArticleGoogle Scholar
- Mondal N, Zhang Y, Jonsson Z, Dhar SK, Kannapiran M, Parvin JD: Elongation by RNA polymerase II on chromatin templates requires topoisomerase activity. Nucleic Acids Res. 2003, 31: 5016-5024. 10.1093/nar/gkg705.PubMedPubMed CentralView ArticleGoogle Scholar
- Desai SD, Li TK, Rodriguez-Bauman A, Rubin EH, Liu LF: Ubiquitin/26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells. Cancer Res. 2001, 61: 5926-5932.PubMedGoogle Scholar
- Carballo M, Gine R, Santos M, Puigdomenech P: Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays. Plant Mol Biol. 1991, 16: 59-70. 10.1007/BF00017917.PubMedView ArticleGoogle Scholar
- De Jong AJ, Hoeberichts FA, Yakimova ET, Maximova E, Woltering EJ: Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta. 2000, 211: 656-662. 10.1007/s004250000341.PubMedView ArticleGoogle Scholar
- Roa H, Lang J, Culligan KM, Keller M, Holec S, Cognat V, Montane MH, Houlne G, Chaboute ME: Ribonucleotide reductase regulation in response to genotoxic stress in Arabidopsis. Plant Physiol. 2009, 151: 461-471. 10.1104/pp.109.140053.PubMedPubMed CentralView ArticleGoogle Scholar
- Deng C, Brown JA, You D, Brown JM: Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae. Genetics. 2005, 170: 591-600. 10.1534/genetics.104.028795.PubMedPubMed CentralView ArticleGoogle Scholar
- Aleksandrushkina NI, Vanyushin BF: Endonucleases and Their Involvement in Plant Apoptosis. Russian Journal of Plant Physiology. 2009, 56: 291-305. 10.1134/S1021443709030017.View ArticleGoogle Scholar
- Sugiyama M, Ito J, Aoyagi S, Fukuda H: Endonucleases. Plant Mol Biol. 2000, 44: 387-397. 10.1023/A:1026504911786.PubMedView ArticleGoogle Scholar
- Giuliani C, Consonni G, Gavazzi G, Colombo M, Dolfini S: Programmed cell death during embryogenesis in maize. Ann Bot. 2002, 90: 287-292. 10.1093/aob/mcf173.PubMedPubMed CentralView ArticleGoogle Scholar
- Bastida M, Graziano E, Roca R, Lopez I, Sanchez-Pons N, Puigdomenech P, Vicient CM: A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death. Planta. 2006, 223: 755-768. 10.1007/s00425-005-0136-5.PubMedView ArticleGoogle Scholar
- Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK: Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J. 2004, 38: 366-379. 10.1111/j.1365-313X.2004.02051.x.PubMedView ArticleGoogle Scholar
- Markmann-Mulisch U, Wendeler E, Zobell O, Schween G, Steinbiss HH, Reiss B: Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair. Plant Cell. 2007, 19: 3080-3089. 10.1105/tpc.107.054049.PubMedPubMed CentralView ArticleGoogle Scholar
- Li J, Harper LC, Golubovskaya I, Wang CR, Weber D, Meeley RB, McElver J, Bowen B, Cande WZ, Schnable PS: Functional analysis of maize RAD51 in meiosis and double-strand break repair. Genetics. 2007, 176: 1469-1482. 10.1534/genetics.106.062604.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen IP, Haehnel U, Altschmied L, Schubert I, Puchta H: The transcriptional response of Arabidopsis to genotoxic stress - a high-density colony array study (HDCA). Plant J. 2003, 35: 771-786. 10.1046/j.1365-313X.2003.01847.x.PubMedView ArticleGoogle Scholar
- Fanning E, Klimovich V, Nager AR: A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res. 2006, 34: 4126-4137. 10.1093/nar/gkl550.PubMedPubMed CentralView ArticleGoogle Scholar
- Malavazi I, Savoldi M, Di Mauro SM, Menck CF, Harris SD, Goldman MH, Goldman GH: Transcriptome analysis of Aspergillus nidulans exposed to camptothecin-induced DNA damage. Eukaryot Cell. 2006, 5: 1688-1704. 10.1128/EC.00167-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Schommer C, Beven A, Lawrenson T, Shaw P, Sablowski R: AHP2 is required for bivalent formation and for segregation of homologous chromosomes in Arabidopsis meiosis. Plant J. 2003, 36: 1-11. 10.1046/j.1365-313X.2003.01850.x.PubMedView ArticleGoogle Scholar
- Enomoto R, Kinebuchi T, Sato M, Yagi H, Kurumizaka H, Yokoyama S: Stimulation of DNA strand exchange by the human TBPIP/Hop2-Mnd1 complex. J Biol Chem. 2006, 281: 5575-5581.PubMedView ArticleGoogle Scholar
- Culligan KM, Robertson CE, Foreman J, Doerner P, Britt AB: ATR and ATM play both distinct and additive roles in response to ionizing radiation. Plant J. 2006, 48: 947-961. 10.1111/j.1365-313X.2006.02931.x.PubMedView ArticleGoogle Scholar
- Dean PJ, Siwiec T, Waterworth WM, Schlogelhofer P, Armstrong SJ, West CE: A novel ATM-dependent X-ray-inducible gene is essential for both plant meiosis and gametogenesis. Plant J. 2009, 58: 791-802. 10.1111/j.1365-313X.2009.03814.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Vignard J, Siwiec T, Chelysheva L, Vrielynck N, Gonord F, Armstrong SJ, Schlogelhofer P, Mercier R: The interplay of RecA-related proteins and the MND1-HOP2 complex during meiosis in Arabidopsis thaliana. PLoS Genet. 2007, 3: 1894-1906.PubMedView ArticleGoogle Scholar
- Carrozza MJ, Utley RT, Workman JL, Côté J: The diverse functions of histone acetyltransferase complexes. Trends Genet. 2003, 19: 321-329. 10.1016/S0168-9525(03)00115-X. ReviewPubMedView ArticleGoogle Scholar
- Bang DD, Ketting R, de Ruijter M, Brandsma JA, Verhage RA, van de Putte P, Brouwer J: Cloning of Schizosaccharomyces pombe rph16+, a gene homologous to the Saccharomyces cerevisiae RAD16 gene. Mutat Res. 1996, 364: 57-71.PubMedView ArticleGoogle Scholar
- Watanabe Y: Shugoshin: guardian spirit at the centromere. Curr Opin Cell Biol. 2005, 17: 590-595. 10.1016/j.ceb.2005.10.003.PubMedView ArticleGoogle Scholar
- Wittmann T, Wilm M, Karsenti E, Vernos I: TPX2, A novel xenopus MAP involved in spindle pole organization. J Cell Biol. 2000, 149: 1405-1418. 10.1083/jcb.149.7.1405.PubMedPubMed CentralView ArticleGoogle Scholar
- Vos JW, Pieuchot L, Evrard JL, Janski N, Bergdoll M, de Ronde D, Perez LH, Sardon T, Vernos I, Schmit AC: The plant TPX2 protein regulates prospindle assembly before nuclear envelope breakdown. Plant Cell. 2008, 20: 2783-2797. 10.1105/tpc.107.056796.PubMedPubMed CentralView ArticleGoogle Scholar
- Batoko H, Moore I: Plant cytokinesis: KNOLLE joins the club. Curr Biol. 2001, 11: R423-426. 10.1016/S0960-9822(01)00251-2.PubMedView ArticleGoogle Scholar
- Peterman TK, Ohol YM, McReynolds LJ, Luna EJ: Patellin1, a novel Sec14-like protein, localizes to the cell plate and binds phosphoinositides. Plant Physiol. 2004, 136: 3080-3094. 10.1104/pp.104.045369.PubMedPubMed CentralView ArticleGoogle Scholar
- Scofield S, Murray JA: KNOX gene function in plant stem cell niches. Plant Mol Biol. 2006, 60: 929-946. 10.1007/s11103-005-4478-y.PubMedView ArticleGoogle Scholar
- Wen Y, Eng CH, Schmoranzer J, Cabrera-Poch N, Morris EJ, Chen M, Wallar BJ, Alberts AS, Gundersen GG: EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat Cell Biol. 2004, 6: 820-830. 10.1038/ncb1160.PubMedView ArticleGoogle Scholar
- Kim JH, Choi D, Kende H: The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis. Plant J. 2003, 36: 94-104. 10.1046/j.1365-313X.2003.01862.x.PubMedView ArticleGoogle Scholar
- Kessler S, Townsley B, Sinha N: L1 division and differentiation patterns influence shoot apical meristem maintenance. Plant Physiol. 2006, 141: 1349-1362. 10.1104/pp.105.076075.PubMedPubMed CentralView ArticleGoogle Scholar
- Mathieu-Rivet E, Gévaudant F, Sicard A, Salar S, Do PT, Mouras A, Fernie AR, Gibon Y, Rothan C, Chevalier C, Hernould M: Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato. Plant J. 2010, 62: 727-741. 10.1111/j.1365-313X.2010.04198.x.PubMedView ArticleGoogle Scholar
- Yu LR, Johnson MD, Conrads TP, Smith RD, Morrison RS, Veenstra TD: Proteome analysis of camptothecin-treated cortical neurons using isotope-coded affinity tags. Electrophoresis. 2002, 23: 1591-1598. 10.1002/1522-2683(200206)23:11<1591::AID-ELPS1591>3.0.CO;2-#.PubMedView ArticleGoogle Scholar
- Ahsan N, Lee DG, Lee KW, Alam I, Lee SH, Bahk JD, Lee BH: Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach. Plant Physiol Biochem. 2008, 46: 1062-1070. 10.1016/j.plaphy.2008.07.002.PubMedView ArticleGoogle Scholar
- Campo S, Carrascal M, Coca M, Abian J, San Segundo B: The defense response of germinating maize embryos against fungal infection: a proteomics approach. Proteomics. 2004, 4: 383-396. 10.1002/pmic.200300657.PubMedView ArticleGoogle Scholar
- Hancock JT, Henson D, Nyirenda M, Desikan R, Harrison J, Lewis M, Hughes J, Neill SJ: Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis. Plant Physiol Biochem. 2005, 43: 828-835. 10.1016/j.plaphy.2005.07.012.PubMedView ArticleGoogle Scholar
- Riccardi F, Gazeau P, de Vienne D, Zivy M: Protein changes in response to progressive water deficit in maize. Quantitative variation and polypeptide identification. Plant Physiol. 1998, 117: 1253-1263. 10.1104/pp.117.4.1253.PubMedPubMed CentralView ArticleGoogle Scholar
- Salekdeh GH, Siopongco J, Wade LJ, Ghareyazie B, Bennett J: Proteomic analysis of rice leaves during drought stress and recovery. Proteomics. 2002, 2: 1131-1145. 10.1002/1615-9861(200209)2:9<1131::AID-PROT1131>3.0.CO;2-1.PubMedView ArticleGoogle Scholar
- Gadjev I, Stone JM, Gechev TS: Programmed cell death in plants: new insights into redox regulation and the role of hydrogen peroxide. Int Rev Cell Mol Biol. 2008, 270: 87-144.PubMedView ArticleGoogle Scholar
- Cohen AA, Geva-Zatorsky N, Eden E, Frenkel-Morgenstern M, Issaeva I, Sigal A, Milo R, Cohen-Saidon C, Liron Y, Kam Z, Cohen L, Danon T, Perzov N, Alon U: Dynamic proteomics of individual cancer cells in response to a drug. Science. 2008, 322: 1511-1516. 10.1126/science.1160165.PubMedView ArticleGoogle Scholar
- Didierjean L, Frendo P, Nasser W, Genot G, Marivet J, Burkard G: Heavy-metal-responsive genes in maize: identification and comparison of their expression upon various forms of abiotic stress. Planta. 1996, 199: 1-8.PubMedView ArticleGoogle Scholar
- Rakwal R, Agrawal GK, Yonekura M: Separation of proteins from stressed rice (Oryza sativa L.) leaf tissues by two-dimensional polyacrylamide gel electrophoresis: induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride. Electrophoresis. 1999, 20: 3472-3478. 10.1002/(SICI)1522-2683(19991101)20:17<3472::AID-ELPS3472>3.0.CO;2-0.PubMedView ArticleGoogle Scholar
- Lee K, Zhan X, Gao J, Qiu J, Feng Y, Meganathan R, Cohen SN, Georgiou G: RraA. a protein inhibitor of RNase E activity that globally modulates RNA abundance in E. coli. Cell. 2003, 114: 623-634. 10.1016/j.cell.2003.08.003.PubMedView ArticleGoogle Scholar
- Yeom JH, Go H, Shin E, Kim HL, Han SH, Moore CJ, Bae J, Lee K: Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA. FEMS Microbiology Letters. 2008, 285: 10-15. 10.1111/j.1574-6968.2008.01205.x.PubMedView ArticleGoogle Scholar
- Cordin O, Banroques J, Tanner NK, Linder P: The DEAD-box protein family of RNA helicases. Gene. 2006, 367: 17-37.PubMedView ArticleGoogle Scholar
- Vashisht AA, Tuteja N: Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance. J Photochem Photobiol B. 2006, 84: 150-160. 10.1016/j.jphotobiol.2006.02.010.PubMedView ArticleGoogle Scholar
- Lorkovic ZJ: Role of plant RNA-binding proteins in development, stress response and genome organization. Trends Plant Sci. 2009, 14: 229-236. 10.1016/j.tplants.2009.01.007.PubMedView ArticleGoogle Scholar
- Cléry A, Blatter M, Allain FHT: RNA recognition motifs: boring? Not quite. Curr Op Struc Biol. 2008, 18: 290-298. 10.1016/j.sbi.2008.04.002.View ArticleGoogle Scholar
- Thompson JE, Hopkins MT, Taylor C, Wang TW: Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development. Trends Plant Sci. 2004, 9: 174-179. 10.1016/j.tplants.2004.02.008.PubMedView ArticleGoogle Scholar
- Hopkins MT, Lampi Y, Wang TW, Liu Z, Thompson JE: Eukaryotic translation initiation factor 5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis. Plant Physiol. 2008, 148: 479-489. 10.1104/pp.108.118869.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu Z, Duguay J, Ma F, Wang TW, Tshin R, Hopkins MT, McNamara L, Thompson JE: Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana. J Exp Bot. 2008, 59: 939-950. 10.1093/jxb/ern017.PubMedView ArticleGoogle Scholar
- Gygi SP, Rochon Y, Franza BR, Aebersold R: Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 1999, 19: 1720-1730.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen G, Gharib TG, Huang CC, Taylor JMG, Misek DE, Kardia SLR, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG: Discordant Protein and mRNA Expression in Lung Adenocarcinomas. Mol Cell Proteomics. 2002, 1: 304-313. 10.1074/mcp.M200008-MCP200.PubMedView ArticleGoogle Scholar
- Tian Q, Stepaniants SB, Mao M, Weng L, Feetham MC, Doyle MJ, Yi EC, Dai H, Thorsson V, Eng J, Goodlett D, Berger JP, Gunter B, Linseley PS, Stoughton RB, Aebersold R, Collins SJ, Hanlon WA, Hood LE: Integrated Genomic and Proteomic Analyses of Gene Expression in Mammalian Cells. Molecular & Cellular Proteomics. 2004, 3: 960-969. 10.1074/mcp.M400055-MCP200.View ArticleGoogle Scholar
- Marti TM, Fleck O: DNA repair nucleases. Cell Mol Life Sci. 2004, 61: 336-354. 10.1007/s00018-003-3223-4.PubMedView ArticleGoogle Scholar
- Tsao YP, D'Arpa P, Liu LF: The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B. Cancer Res. 1992, 52: 1823-1829.PubMedGoogle Scholar
- Zhou Y, Gwadry FG, Reinhold WC, Miller LD, Smith LH, Scherf U, Liu ET, Kohn KW, Pommier Y, Weinstein JN: Transcriptional regulation of mitotic genes by camptothecin-induced DNA damage: microarray analysis of dose- and time-dependent effects. Cancer Res. 2002, 62: 1688-1695.PubMedGoogle Scholar
- Zhou FF, Yan M, Guo GF, Wang F, Qiu HJ, Zheng FM, Zhang Y, Liu Q, Zhu XF, Xia LP: Knockdown of eIF4E suppresses cell growth and migration, enhances chemosensitivity and correlates with increase in Bax/Bcl-2 ratio in triple-negative breast cancer cells. Med Oncol. 2011.Google Scholar
- Kunz BA, Cahill DM, Mohr PG, Osmond MJ, Vonarx EJ: Plant responses to UV radiation and links to pathogen resistance. Int Rev Cytol. 2006, 255: 1-40.PubMedView ArticleGoogle Scholar
- Hack CJ: Integrated transcriptome and proteome data: the challenges ahead. Brief Funct Genomic Proteomic. 2004, 3: 212-219. 10.1093/bfgp/3.3.212.PubMedView ArticleGoogle Scholar
- Jiang Y, Yang B, Harris NS, Deyholos MK: Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J Exp Bot. 2007, 58: 3591-36007. 10.1093/jxb/erm207.PubMedView ArticleGoogle Scholar
- Yan SP, Zhang QY, Tang ZC, Su WA, Sun WN: Comparative proteomic analysis provides new insights into chilling stress responses in rice. Mol Cell Proteomics. 2006, 5: 484-496.PubMedView ArticleGoogle Scholar
- Olivares-Hernández R, Bordel S, Nielsen J: Codon usage variability determines the correlation between proteome and transcriptome fold changes. BMC Syst Biol. 2011, 25: 33-View ArticleGoogle Scholar
- Cagas SE, Jain MR, Li H, Perlin DS: Profiling the Aspergillus fumigatus proteome in response to caspofungin. Antimicrob Agents Chemother. 2011, 55: 146-154. 10.1128/AAC.00884-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Anderson P, Kedersha N: RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol. 2009, 10: 430-436.PubMedView ArticleGoogle Scholar
- Smalle J, Vierstra RD: The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol. 2004, 55: 555-590. 10.1146/annurev.arplant.55.031903.141801.PubMedView ArticleGoogle Scholar
- Lin CP, Ba Y, Lyu YL, Desai SD, Liu LF: A ubiquitin-proteasome pathway for the repair of topoisomerase I-DNA covalent complexes. J Biol Chem. 2008, 283: 21074-21083. 10.1074/jbc.M803493200.PubMedPubMed CentralView ArticleGoogle Scholar
- Yoshida N, Yanai Y, Chen L, Kato Y, Hiratsuka J, Miwa T, Sung ZR, Takahashi S: EMBRYONIC FLOWER2, a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis. Plant Cell. 2001, 13: 2471-2481.PubMedPubMed CentralView ArticleGoogle Scholar
- Goda H, Sasaki E, Akiyama K, Maruyama-Nakashita A, Nakabayashi K, Li W, Ogawa M, Yamauchi Y, Preston J, Aoki K, Kiba T, Takasuto S, Fujioka S, Asami T, Nakano T, Kato H, Mizuno T, Sakakibara H, Yamaguchi S, Nambara E, Kamiya Y, Takahashi H, Hirai MY, Sakurai T, Shinozaki K, Saito K, Yoshida S, Shimada Y: The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access. Plant J. 2008, 55: 526-542. 10.1111/j.1365-313X.2008.03510.x.PubMedView ArticleGoogle Scholar
- Olsen AN, Ernst HA, Leggio LL, Skriver K: NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci. 2005, 10: 79-87. 10.1016/j.tplants.2004.12.010.PubMedView ArticleGoogle Scholar
- Bannenberg G, Martinez M, Rodriguez MJ, Lopez MA, Ponce de Leon I, Hamberg M, Castresana C: Functional analysis of alpha-DOX2, an active alpha-dioxygenase critical for normal development in tomato plants. Plant Physiol. 2009, 151: 1421-1432. 10.1104/pp.109.145094.PubMedPubMed CentralView ArticleGoogle Scholar
- Mascarell-Creus A, Canizares J, Vilarrasa-Blasi J, Mora-Garcia S, Blanca J, Gonzalez-Ibeas D, Saladie M, Roig C, Deleu W, Pico-Silvent B, Lopez-Bigas N, Aranda MA, Garcia-Mas J, Nuez F, Puigdomenech P, Cano-Delgado AI: An oligo-based microarray offers novel transcriptomic approaches for the analysis of pathogen resistance and fruit quality traits in melon (Cucumis melo L.). BMC Genomics. 2009, 10: 467-10.1186/1471-2164-10-467.PubMedPubMed CentralView ArticleGoogle Scholar
- Thelen MP, Northcote DH: Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis. Planta. 1989, 179: 181-195. 10.1007/BF00393688.PubMedView ArticleGoogle Scholar
- Irar S, Brini F, Goday A, Masmoudi K, Pagès M: Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) -- A wider perspective of the proteome. J Proteomics. 2010, 73: 1707-1721. 10.1016/j.jprot.2010.05.003.PubMedView ArticleGoogle Scholar
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