134Caesium uptake and morphology of plants
A. thaliana Col-0 were grown for 5 wk on a medium containing 0.7 mm K+ and very low concentrations of 134Cs (1.5–9.4 × 10−12 m). Including the inactive Cs, the total Cs concentration was 1.3–7.9 × 10−10 m. These concentrations were far below the known phytotoxic concentrations of 133Cs, which range from 5 × 10−4 m to 7 × 10−4 m (Sahr et al., 2005). Therefore, all effects observed upon 134Cs application were caused by low chronic doses of ionizing radiation and not by the known effects of Cs ions (Hampton et al., 2004; Sahr et al., 2005). Increasing the external activity of 134Cs resulted in an increased accumulation of 134Cs in leaves, as measured by an increased activity of up to 1 Bq mg−1 fresh weight (f. wt) in the leaves (Fig. 1). The absorbed dose rate of an Arabidopsis plant for external γ-radiation and for internal β- and γ-radiation exposure, as a result of 134Cs, was estimated by using the dose rate conversion coefficients, calculated for reference terrestrial biota, of Taranenko et al. (2004) and Gómez-Ros et al. (2004). The dose rate of the external exposure was only approx. 0.1 µGy h−1 and, thus, negligibly small as compared to the dose rate of the internal exposure amounting to 50–100 µGy h−1. The internal and external exposure caused by natural radionuclides, mainly 40K, was < 1 µGy h−1. Thus, the absorbed dose rate of an Arabidopsis plant was dominated by the internal exposure to 134Cs and higher than the natural background by a factor of approx. 100, but smaller, by several orders of magnitude, than the dose rate caused by γ-irradiation in many other studies. Real et al. (2004), reviewing all data available on the effects induced by ionizing radiation in various wildlife groups, conclude that the threshold for statistically significant effects is a dose rate of approx. 100 µGy h−1.
Figure 1. 134Caesium activity in Arabidopsis thaliana Col-0. Plants were grown for 5 wk in the absence or presence of 133CsCl (100 µm) and in the indicated activities of 134Cs. Error bars ± standard deviation (SD) (n = 3) are shown for each data point. f. wt, fresh weight.
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The internal 134Cs contamination in A. thaliana was much higher than in plants grown on different soil types that were spiked with 134CsCl (Zaka et al., 2002; Tang & Willey, 2003). Although Cs accumulation varies in different plant species (Broadley & Willey, 1997; Tang & Willey, 2003; White et al., 2003), the high 134Cs accumulation in this study was mainly dependent on an increased bioavailability using agar medium. Application of 100 µm 133Cs resulted in a further slight uptake of 134Cs (Fig. 1). This is in accordance with an increased uptake of Cs caused by increasing the external 133/137Cs concentration (Fuhrmann et al., 2002; White et al., 2003; Hampton et al., 2004; Sahr et al., 2005). Up to an activity of 30 Bq cm−3, no influence on morphology and development of plants was found. External activities of 60 Bq cm−3 resulted in a growth reduction of leaves, stem and roots. However, flowering and seed production was not affected at all.
Isolation of genes up- and down-regulated by 134Cs
Abiotic and biotic stress responses, detected by an increased or a decreased level of transcripts, are often not specific, as identical genes might be affected also by other stress factors (Heidenreich et al., 1999; Reymond et al., 2000; Broschéet al., 2002; Inzé & Van Montagu, 2002; and articles therein; Mahalingam et al., 2003). In addition, the dose, as well as the duration of stress given, might affect the stress specificity. Focussing on ionizing radiation, gene-expression profiling of human carcinoma cells revealed overlapping and distinct classes of genes, both of which are induced by internal β- and by external γ-radiation (Marko et al., 2003). Recently, we described the isolation of 133Cs-affected genes in roots of Arabidopsis by SSH (Sahr et al., 2005). To compare these 133Cs effects with the low level of ionizing radiation of 134Cs at the transcriptional level, Arabidopsis plants were grown for 5 wk in a hydroponic medium containing 134CsCl at an activity of 30 Bq cm−3. This value is comparable with realistic outdoor activities in the Chernobyl zone (White & Broadley, 2000). Although 134Cs was incorporated into the plants we cannot distinguish between external and internal radiation effects caused by the high β-energy of 2.059 MeV and the additional γ-radiation, although the dose rate of the internal exposure was much higher than the external exposure.
Clones obtained by the SSH were further analysed by DNA-microarray analysis and only clones that showed differences in the signal intensities (subtracted vs nonsubtracted) with a factor of > 2 or < 0.5 were taken as positive. After sequencing, redundant clones were eliminated that finally resulted in 46 annotated genes (Tables 1 and 2). These genes were grouped into nine classes according to their function (Table 1). The slight discrepancy between the numbers of genes in Table 1 (50) vs the 46 134Cs-affected genes was a result of double designation, caused by an unclear assignment. Out of the 46 influenced genes, 41 were up-regulated and five were down-regulated (Table 2). As a control for the validity of the microarray data, RT–PCR amplification was carried out for selected genes (Table 3). Arabidopsis plants were grown in the presence of 134Cs (10, 20 and 60 Bq cm−3) and roots were harvested after 5 wk. Duplicate biological samples and up to six independent RT–PCR amplifications for each sample were carried out in order to verify even very weakly induced or repressed genes, respectively (Table 3).
Table 1. Composition of clusters of the isolated 134Cs-affected expressed sequence tags (ESTs) in roots of Arabidopsis thaliana
|Major functional categories||Up||Down|
|Cellular metabolism|| 3||2|
|Cell growth, division and development|| 7||–|
|Transcription and translation|| 3||1|
|Protein synthesis, folding and modification|| 1||–|
|Transport and homeostasis|| 1||–|
|Cellular communication and signalling|| 2||–|
|Defence, stress response and detoxification||10||–|
Table 2. Up- and down-regulated (marked in italics) transcripts in roots of Arabidopsis thaliana, isolated by using the suppression subtractive hybridization (SSH) method
|At3g60245||60S ribosomal protein L37A like|
|At3g59540||60S ribosomal protein L38-like protein|
|At1g21400||Alpha ketoacid-dehydrogenase E1 alpha subunit|
|At5g47120||Bax inhibitor-1 like|
|At1g20620||Unknown protein, catalase signatures|
|At5g41770||Cell cycle control crn protein-like|
|At5g21900||DNA excision repair protein|
|At4g30650||Low temperature and salt responsive protein homolog|
|At5g06150||Mitosis-specific cyclin 1b|
|At4g00680||Putative actin-depolymerizing factor|
|At4g38250||Putative amino acid transporter protein|
|At2g33830||Putative auxin-regulated protein|
|At2g05510||Putative glycine-rich protein|
|At4g23670||Putative major latex protein|
|At1g65980||Unknown putative protein|
|At3g48530||Unknown putative protein|
|At3g10860||Putative ubiquinol-cytochrome c reductase complex ubiquinon-binding protein|
|At2g33770||Putative ubiquitin conjugating enzyme E2-like protein|
|At3g56020||Ribosomal protein GL41-like|
|At1g64660||Similar to O-succinylhomoserine sulfhydrylase|
|At5g15970/60||Stress inducible kin2/kin1 protein|
|At5g19780||Tubulin alpha-5 chain|
|At3g14100||Oligouridylate binding-like protein|
|At5g07440||Glutamate dehydrogenase 2|
Table 3. Up-regulated expressed sequence tags (ESTs) in roots of Arabidopsis thaliana, isolated by the suppression subtractive hybridization (SSH) method and verified by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) and real-time RT–PCR
|Accession number||Annotation||Semiquantitative RT–PCR||Quantitative real-time RT–PCR|
|At5g41770||Cell cycle control crn protein-like||1.4 (0.1)||1.2 (0.001)|
|At5g06150||Mitosis-specific cyclin 1b||1.5 (0.02)||2.3 (0.001)|
|At4g29350||Profilin 2||1.5 (0.08)||1.6 (0.001)|
|At5g19780||Tubulin α-5 chain||1.6 (0.08)||1.2 (0.001)|
|At2g33830||Putative auxin-regulated protein||1.8 (0.07)||2.3 (0.001)|
|At5g21900||DNA excision repair protein||1.4 (0.003)||1.1 (0.001)|
|At5g42980||Thioredoxin||1.5 (0.003)||1.9 (0.001)|
|At5g15970/60||Cold-regulated protein (kin2/kin1)||1.6 (0.008)||1.4 (0.5)|
Functional classification of the isolated genes
One group of induced transcripts are involved in general metabolism (Tables 1 and 2), including 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR), O-succinylhomoserine sulfhydrylase and α-ketoacid dehydrogenase. 3-Hydroxy-3-methylglutaryl-CoA-reductase is a key enzyme of isoprenoid biosynthesis that is up-regulated by abiotic and biotic stress factors (Yang et al., 1991; McGarvey & Croteau, 1995). Furthermore, it has been shown that 3-hydroxy-3-methylglutaryl-CoA-synthase, which precedes HMGR, is induced by ozone (Wegener et al., 1997a). As ozone treatment, as well as ionizing radiation, results in the production of reactive oxygen species (ROS) (Riley, 1994; Langebartels et al., 2002; Rugo & Schiestl, 2004; Streffer et al., 2004), this might be a common link of these stress factors. Two other transcripts of this group, a glutamate dehydrogenase and xylose isomerase, important for nitrogen and carbohydrate metabolism, were down-regulated with 134Cs stress (Table 2).
An important group of up-regulated mRNAs are involved in cell growth, cell division and development (Tables 1 and 2). Transcripts of a cell cycle crn control protein-like and of a mitosis-specific cyclin B1 were induced by a factor of 1.2–1.4 and of 1.5–2.3, respectively (Table 3). Cell cycle proteins are involved in the regulation of mitosis and RNA processing in eukaryotes (Preker & Keller, 1998). Together with cyclin-dependent protein kinases, cyclins are essential for cell cycle control (Weingartner et al., 2003). It is also known in plants that substances such as aphidicolin, which damage or disturb the DNA replication, induce the gene expression of cyclin B1 and lead to a G2 arrest (Culligan et al., 2004). The transcriptional response of Arabidopsis to genotoxic stress by the application of bleomycin and mitomycin C resulted also in an up-regulation of repair cell cycle genes (e.g. cyclin-dependent kinases and CycB1) (Chen et al., 2003). In addition, crn-like proteins are also induced upon Cs, salt, osmotic and UV stress, and cyclin B1 has been shown to be induced by heat stress and induced programmed cell death (Swidzinski et al., 2002; Craigon et al., 2004; http://affymetrix.arabidopsis.info/narrays/geneswinger.pl).
Transcripts for profilin 2, a putative actin-depolymerizing factor and α-5 tubulin were also induced by 134Cs (Tables 2 and 3), indicating a reorganization of the plant's cytoskeleton upon ionizing radiation. Profilins are localized in the cytoplasm, as well as in the nucleus, and are regulatory proteins of actin modulation (Christensen et al., 1996). Profilins are also induced upon exposure to several other stresses, such as Cs, salt, osmotic stress and UV radiation (Craigon et al., 2004; Hampton et al., 2004; http://affymetrix.arabidopsis.info/narrays/geneswinger.pl). Microtubules, together with tubulin-associated proteins, regulate root growth and are important for stabilization of the intracellular localization of parts of the cytoplasma and for cell division (Jacobs et al., 1988; Bibikova et al., 1999). Cyclin B1 has been shown to be localized to microtubules in human cells (Jackmann et al., 1995). This suggests that an up-regulation of α-5 tubulin and cyclin B1 transcripts upon ionizing radiation contributes to functional microtubules.
Transcript levels of two 60S ribosomal protein transcripts and of a G41-like ribosomal protein were increased upon exposure to 134Cs (Table 2). Ribosomal proteins, important for protein synthesis, are also induced upon genotoxic stress (Chen et al., 2003) or UVB radiation (Ernst et al., 2001; Izaguirre et al., 2003; Casati & Walbot, 2004). Gamma-radiation of human carcinoma cells also resulted in an increased amount of a 40S ribosomal protein and a ribosomal S6 kinase (Marko et al., 2003). This indicates that protein synthesis in plant cells is sensitive to low ionizing radiation and not only to UV radiation.
A putative ubiquitin-conjugating enzyme E2 was up-regulated by 134Cs (Table 2). Similarly, a ubiquitin-conjugating enzyme E2 has also been shown to be induced by genotoxic stress (Chen et al., 2003). Ozone fumigation of Scots pine seedlings was found to result in the accumulation of polyubiquitin mRNA (Wegener et al., 1997b), indicating again the involvement of ROS in signalling, at least partially, the effects of ionizing radiation. In addition to the well-known function of ubiquitins in the degradation of damaged proteins, the ubiquitin system is also of importance in DNA repair, by modification of involved enzymes, thus influencing their activities (Hiller et al., 1996; Ciechanover, 1998; Hellmann & Estelle, 2002; Pickart, 2002).
Only two transcripts for cellular communication and signal transduction were found to be induced by 134Cs: a bax inhibitor-1 like protein and a putative auxin-regulated protein (Tables 2 and 3). The bax inhibitor-1 protein is a mammalian apoptosis suppressor and the auxin-regulated protein might have protein kinase activity (Xu & Reed, 1998; Müllauer et al., 2001). Putative auxin-regulated proteins are also induced upon exposure to several other factors such as Cs, salt, osmotic stress and UV radiation (Craigon et al., 2004; Hampton et al., 2004; http://affymetrix.arabidopsis.info/narrays/geneswinger.pl). Recently, it has been shown that an expressed sequence tag of this putative auxin-regulated protein (At2g33830) was up-regulated in A. thaliana by bacterial colonization (Cartieaux et al., 2003). Auxin, an important plant growth regulator, is associated with cell division, growth, maturation and cell differentiation (Trewavas, 2000). Auxin is known to regulate gene expression via protein degradation, including the ubiquitin system (Dharmasiri & Estelle, 2004; Weijers & Jürgens, 2004), and a putative ubiquitin-conjugating enzyme E2-like protein mRNA (At2g33770) was induced upon 134Cs application (Table 2).
Genes coding for cell defence, stress response and detoxification (Table 1) include a DNA-excision repair protein with an induction factor of up to 1.4 (Table 3). DNA-excision repair proteins are involved in the recognition of DNA single-strand breakages that will be excised and then replaced. In addition, we analyzed the Mre11 protein transcript that is part of the DNA recombination complex, important for the repair of DNA double-strand breakages (Paull & Gellert, 1998). Semiquantitative RT–PCR of Mre11 protein transcripts resulted in a significant induction factor of 1.3 (P = 0.03) with a 134Cs activity of 60 Bq cm−3. The importance of genes involved in DNA double-strand repair has been shown in Arabidopsis mutants that were sensitive to γ-radiation (Hefner et al., 2003). The up-regulation of thioredoxin and catalase mRNAs (Tables 2 and 3) indicates again the involvement of oxidative stress and the production of ROS following treatment with 134Cs. Interestingly, the identical thioredoxin gene was also up-regulated following treatment with bleomycin plus mitomycin C (Chen et al., 2003). The idea of ROS production is further supported by semiquantitative RT–PCR for superoxide dismutase (SOD) transcripts that resulted in the up-regulation of ROS transcripts by a factor of 1.4 (P = 0.02). For SOD and catalase transcripts, an ozone-induced accumulation is well known (Langebartels et al., 2002). Similarly, small heat shock proteins were induced upon oxidative stress and γ-radiation in tomato, as well as by ozone treatment of parsley plants (Eckey-Kaltenbach et al., 1997; Banzet et al., 1998). In addition, the induction of an extensin-like protein transcript (Table 2) was also found upon ozone treatment of Scots pine, Norway spruce and European beech (Schneiderbauer et al., 1995). Finally, genes involved in temperature and salt stress were also induced by 134Cs (Table 2; Gong et al., 2001; Xiong et al., 2002). In a latter group, genes are summarized that cannot exactly be classified.