Growth of plants before stress treatments
The conceptual aim of our study was to generate a sufficiently complete and time-resolved transcriptome data set to enable accurate bioinformatics determination of abiotic stress-induced gene expression in Arabidopsis. Although global transcriptional data sets are available, this approach has been limited due to differences in plant growth conditions (e.g. age of plants, light regime, growth media), stress application (e.g. transient versus continuous), harvested tissues, type of microarray used (cDNA chip, AG1, ATH1) and statistical and bioinformatics criteria (for details see Supplementary).
The methodical background of our cultivation conditions was to grow healthy plants in a way that excluded any biotic and abiotic stress and to produce a sufficient amount of root and shoot tissue for efficient extraction of RNA. Therefore, we avoided growing plants in soil or on agar plates. Firstly, the time-consuming harvesting of clean roots out of soil or agar would have induced enormous wounding and water stress in the tissue. Secondly, the application of solutions to soil-grown plants in a spatio-temporally reproducible manner is almost impossible due to the non-homogeneous nature of soil density. We therefore decided to initially cultivate the plants for 13 days at 24°C under sterile conditions on polypropylene rafts in growth boxes containing MS medium supplemented with 0.5% agar and 0.5% sucrose (Figure 1a). Preceding tests of different cultivation procedures had revealed that the addition of sucrose to the medium was necessary during the initial incubation phase for successful cultivation of Arabidopsis. The boxes were closed with a lid containing an opening for air ventilation. During growth the plants were kept under long-day conditions (16 h light/8 h dark) at a light intensity of 150 µmol photons m−2 sec−1 and a relative humidity of 50% in a standard phytochamber. Thirteen days after sowing, the plant-containing rafts (Figure 1b) were transferred to new growth boxes containing fresh liquid MS medium without sucrose and were cultivated for five additional days under the described conditions. At this time, the plants had developed sufficient amounts of root and shoot tissue but had not initiated flowering. From several different growth regimes tested, the described growth arrangement was chosen because it ensured the most reproducible and comparable growth conditions.
Figure 1. Principle of the cultivation of Arabidopsis plants for the abiotic stress treatments. (a) Schematic representation of a cultivation vessel used for the growth of Arabidopsis plants. (b) Representative picture of Arabidopsis plants after 18 days of cultivation. Culturing the plants on floating membrane rafts enabled simple exchange of the media and application of substances as well as fast harvest of the shoot and root tissue.
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The stress treatments were initiated in parallel 18 days after sowing and 3 h after dark/light transition. The parallel performance of all stress treatments excluded differential circadian effects which could superimpose stress-induced gene expression. For stress treatments, the plants were carefully transferred from the standard phytochamber to the laboratory where all treatments were performed and then returned to the growth chamber until harvesting. For harvesting, the plants were removed from the raft and the roots were cut off. From one box only either the shoot or root material of nine plants was harvested and pooled to avoid handling stress in the other respective tissue and to minimize physiological differences between single plants. The entire harvest procedure was completed within less than 10 min. Root and shoot samples were taken in two biological replicas 0 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after the onset of stress treatment. In cases of expected very fast stress-induced transcriptional alterations (e.g. UV-B light) samples were also harvested 15 min after the onset of stress application. The RNA samples for control kinetics were generated from non-stressed plants which were handled in exactly the same way but were not exposed to stress conditions.
Cold, heat, drought, osmotic, salt, wounding and UV-B light stress applications were performed as described below.
cRNA hybridisation and data processing
Hybridization of cRNA using the Affymetrix AHT1 gene chip was accomplished according to a modified Affymetrix protocol developed at the Deutsche Ressourcenzentrum für Genomforschung (RZPD) in Berlin, Germany (http://www.rzpd.de). The raw data were imported into specialist and general data processing tools, GeneSpring and R, and were processed and normalized using the algorithms of these program packages (see also Experimental procedures). The correlation between the data of the two biological replicas of the global abiotic stress experiment was never less than 0.95, as in the examples shown for the cold, drought, UV-B light and control data sets (Supplementary). These results demonstrate the very high reliability, reproducibility and quality of the raw data. Therefore, the mean of the results of the two biological replicas are presented in all figures and tables. The accession numbers for the array data at the Arabidopsis Information Resource (TAIR) are provided in Experimental procedures.
General pattern of abiotic stress-regulated gene expression
To identify genes which are contemporaneously regulated by different stimuli we compared the gene expression patterns induced by the applied abiotic stresses.
Comparing the overall number of genes whose expression responded to one of the three treatments over a 24-h period we detected a large difference in the number of differentially regulated genes (Table 1). With the exception of high osmolarity, the number of genes whose expression was upregulated in response to the abiotic stresses exceeded the number of genes downregulated under the respective conditions (Figure 2).
Table 1. Numbers of genes differentially regulated during abiotic stress treatments
| ||Time points, root||Time points, shoot|
|0.25 h||0.5 h||1.0 h||3.0 h||6.0 h||12 h||24 h||0.25 h||0.5 h||1.0 h||3.0 h||6.0 h||12 h||24 h|
|Cold stress upregulated||ND||48||51||212||511||797||1001||ND||33||190||409||716||1030||1314|
|Cold stress downregulated||ND||12||11||55||81||384||826||ND||120||23||89||246||941||1328|
|Drought stress upregulated||110||325||309||32||131||42||6||44||141||218||217||122||129||60|
|Drought stress downregulated||12||42||161||16||58||23||12||59||93||21||39||22||8||14|
|UV-B stress upregulated||45||197||299||286||24||16||201||102||287||534||1125||1262||304||380|
|UV-B stress downregulated||49||40||132||305||21||7||113||55||55||84||528||743||91||70|
|Salt stress upregulated||ND||132||575||1263||1692||793||1310||ND||6||11||361||448||326||735|
|Salt stress downregulated||ND||14||240||421||1291||577||666||ND||27||22||218||187||177||494|
|Osmotic stress upregulated||ND||189||347||429||597||908||788||ND||34||214||797||934||1214||1553|
|Osmotic stress downregulated||ND||24||199||367||525||906||590||ND||23||30||325||760||1345||1832|
|Heat stress upregulated||41||122||468||1139||79||255||13||22||52||395||651||153||63||13|
|Heat stress downregulated||116||40||270||923||98||328||136||269||169||325||787||80||33||39|
|Wound stress upregulated||18||34||152||17||41||181||34||201||388||562||227||164||276||152|
|Wound stress downregulated||22||8||87||24||11||114||19||47||77||103||15||6||27||26|
Figure 2. Total number of genes differentially up- (black bars) and downregulated (white bars) in roots and shoots in response to cold (a), drought (b), UV-B light (c), high salt (d), high osmolarity (e), heat (f) and wounding (g) stress treatment. The tissues for RNA extraction were harvested at the indicated time points. For data normalization, processing and statistical analysis as well as for the classification of genes to be up- or downregulated see Experimental procedures.
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The number of drought-responsive genes in our study is significantly lower than that found in the studies of Kreps et al. (2002) and Seki et al. (2002). Furthermore, the plants recovered from the drought as indicated by the transient pattern of gene expression (Figure 2). This is very likely a result of differences in stress application: Whereas Kreps et al. (2002) and Seki et al. (2002) applied a prolonged drought stress (Supplementary), the treatment in our study was transient, and thus modest. This was reflected in the expression pattern of several genes considered to be marker genes for abiotic stress (Supplementary). Secondly, in the study of Kreps et al. (2002) the drought stress was applied by the incubation of the plants in 200 mm mannitol (Supplementary). This treatment also induces genes specific for osmotic stress (Figure 2; JK, unpublished results).
A comparison of the kinetics of changes in expression patterns in root and shoot tissues revealed that cold and osmotic stress induced a continuous increase in expressed genes whilst, to a different extent, salt, heat, UV-B light, drought and wounding stress caused transient alterations in gene activity (Figure 2). This observation may reflect the nature (constant versus transient) of the stress exposure. Interestingly, the number of genes responsive to drought and wounding upregulated at the 3- to 6-h time point is lower than those at 1 h and 6 h and 12 h, respectively. This indicates a biphasic response of this organ to drought stress and wounding.
Irrespectively of the type of stress applied to the plants, gene expression responded very fast to the altered environmental conditions in both roots and shoots. Significant changes in gene expression were already observed 15 and 30 min after the onset of treatment (Figure 2). Cold treatment affected the expression of a similar number of genes in both root and shoot tissue, respectively. Remarkably, the shoot-expressed genes appeared to respond more intensely over time, although the root potentially cooled down faster due to the way the plants were handled (Figure 2). Ultraviolet-B light and wounding stress were only applied to the aerial parts of the plants. However, these stresses induced fast responses in gene expression not only in the shoot but also in the root (Figure 2). The opposite was observed for salt and osmotic stress, where treatment of the roots caused significant alterations in the shoot (Figure 2).
Common elements of drought-, cold- and UV-B light-regulated gene expression
We then studied drought-, cold- and UV-B light-regulated gene expression in more detail. This analysis was of particular interest because the kind of stress as well as the duration of the stress exposure were different (e.g. continuous, cold; transient, drought, UV-B light). Accordingly, the identification of genes commonly induced during the early response to all three stimuli may point to general and important components involved in stress-related signalling.
Response reactions to abiotic stresses like cold, drought and UV light are likely to share common second messenger molecules like Ca2+ or ROS. We therefore set out to investigate whether there are groups of genes which are either regulated by a specific stimulus or, alternatively, which show a transcriptional response to all stresses investigated. The latter group might represent targets of signalling components/reactions common to all responses to abiotic stresses.
As shown in Figure 3, the majority of up- and downregulated genes were specific for the applied stresses. Nevertheless, the expression of a significant number of genes was upregulated during all three different stress responses, especially at very early time points (Figure 3, Table 3, Supplementary). For example, we found nine genes concertedly upregulated in the shoot 30 min after the onset of the treatment. This number increases to 59 at the 1-h time point (Figure 3). In relation to the total number of differentially expressed genes, the number of concertedly regulated genes decreased with time (Figure 3, Table 3, Supplementary). Furthermore, there appeared to be a tendency that the upregulated genes were shared to a higher degree than the downregulated genes in both roots and shoots (Figure 3). In addition, UV-B light and drought stress treatment affected the expression of a larger number of upregulated genes in a similar way in both organs at early time points (0.5–1.0 h) when compared with the other combinations of stress factors (drought versus cold, UV-B light versus cold; Figure 3). For instance, 0.5 h after stress application drought and UV-B light stress shared 70 upregulated genes in the shoot, whereas the corresponding value were 15 genes for cold/UV-B light and 13 for cold/drought stress combinations.
Figure 3. Specificity and interference of genes regulated in response to cold, drought and UV-B light stress in shoots and roots. The Venn diagrams depict data of all time points after stress application (0.5–24 h). The identity of the genes commonly upregulated in all three stress responses is provided in Table 2.
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Table 2. Percentage of common upregulated genes during cold, drought and UV-B light stress in shoots
| ||Time point|
|0.5 h||1.0 h||3.0 h||6.0 h||12 h||24 h|
|Common upregulated genes (%)||1.9||6.3||0.5||0.7||1.0||0.3|
|Common transcriptional regulators (%)||66.7||35.6||0.0||21.4||26.7||16.7|
Table 3. Percentage of common upregulated genes during UV-B light stress in roots and shoots
| ||Time points|
| ||0.25 h||0.5 h||1.0 h||3.0 h||6.0 h||12 h||24 h|
|Common upregulated genes (%)||7.8||14.4||9.8||4.1||0.6||0.6||3.4|
|Common downregulated genes (%)||10.5||1.1||1.5||1.7||0.0||0.0||0.0|
Rapidly induced genes common to drought, cold and UV-B light stress treatments
We next concentrated our analyses on genes rapidly induced by all stresses in the shoots within the first few hours. These genes might encode plant core environmental stress response (PCESR) proteins with important functions in various stress response pathways as known for Saccharomyces cerevisiae and Schizosaccharomyces pombe (Causton et al., 2001; Chen et al., 2003). Thirty minutes after the onset of stress treatments there was a group of nine genes induced in the whole shoot by all three stresses (Figure 3, Supplementary). The majority of these genes were also immediately responsive to salt and osmotic stress and wounding (Figure 4). At least six of the nine fast-induced genes encode bona fide transcriptional regulators [Figure 4, Supplementary; Arabidopsis transcription factor data base (DATF; http://datf.cbi.pku.edu.cn)]. However, these genes showed differential expression kinetics depending on the applied stress (Figure 4). For instance, the C2H2 zinc finger transcription factor ZAT10 displayed a one-peak, transient expression pattern during UV-B and cold stress, a biphasic profile during drought stress and a sustained expression during wounding (Figure 4).
Figure 4. Overall expression kinetics of the nine genes commonly upregulated in the shoot at the 0.5 h time point after onset of cold, drought, UV-B, salt, osmotic stress treatment and wounding. The identity of the genes is provided at the bottom.
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Within 1 h the number of commonly induced genes increased to 59 (Figure 3). Of these 59 genes, 21 represent transcriptional regulators. In addition to members of the ERF and MYB family these again include several C2H2 zinc finger transcription factors such as AZF2, ZAT10 and ZAT12 (Supplementary). This set of early responding transcription factors was complemented by genes for signalling components such as, for instance, proteins involved in Ca2+ signalling (Supplementary). At later time points the percentage of commonly regulated genes and the contribution of transcriptional regulators decreased significantly (Table 3, Supplementary) suggesting that stress-specific response pathways had been initiated and were proceeding.
We used vector analysis (VA; Breitling et al., 2005) as a means to quantitatively represent the expression dynamics of a single gene under different experimental conditions. Here VA provides an impression of how a given gene might be controlled in the plant during simultaneous exposure to different stresses by theoretical means. We focused on the 59 genes which were concurrently upregulated in the shoot 1 h after induction during cold, drought and UV-B light stress treatment (Figure 3; Supplementary). Because VA enables a two-dimensional representation only, we compared the stresses in pairs (Figure 5). A significant number of the 59 genes were already upregulated 0.5 h after the application of all three stresses (Figure 5a–c). The comparison of cold and UV-B light stress indicated that, at early time points, UV-B light stress slightly dominated over the regulatory influence of cold stress (Figure 5a). At later time points cold was predominant over UV-B light (Figure 4a). At the 12-h time point cold repressed the expression of some genes which were strongly upregulated by UV-B light (and cold) at 1 h. When UV-B light and drought stress were compared, it was recognizable that UV-B dominated over the regulatory influence of drought (Figure 5b). Although drought was slightly predominant over cold at 0.5 h after stress application, the latter had a stronger influence on the expression of the selected genes at later time points (Figure 5c).
Figure 5. Vector analysis (VA) of genes commonly upregulated in the shoot by cold, drought and UV-B light stress. The VA over time (0.5 to 12 h) was carried using the 59 genes (squares) commonly upregulated in the shoot 1 h after onset of the stress treatment according to Breitling et al. (2005). For details see Experimental procedures. (a) Vector analysis of cold versus UV-B light stress. (b) Vector analysis of UV-B light versus drought. (c) Vector analysis of drought versus cold.
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Principal component analysis of stress-induced gene expression
To reveal potential response pathways specific to drought, cold and UV-B light stress, we applied a principal component analysis (PCA; Schoelkopf et al., 1998) using the root and shoot data set. Principal component analysis converts large microarray data sets into a few representative numbers for each sample giving a transcriptional ‘signature’ for each experimental condition. These numbers can be interpreted as a measure of distance between the samples, sacrificing the information on specific genes for a global view. Thus, the closer two samples are in the PCA, the closer the similarity of the overall transcriptional expression.
At the very early time points after onset of stress application the samples clustered together indicating a high similarity in the early transcriptional responses to the different stresses (Figure 6). This suggests that the immediate response of the plant to these stresses is non-specific and contains only minor tissue-specific elements. However, at later time points stress-, tissue- and time-specific responses became apparent. For instance, UV-B light irradiation of the shoots induced a large and distinct transcriptional response which differed significantly from that caused by the other stress treatments (Figure 6). Furthermore, although the roots of UV-B-treated plants clearly responded to the shoot-applied stress, the reaction is not as intense and UV-B light-specific as in the shoot (Figure 5). In contrast, the shoot and root tissue showed a very similar transcriptional response to cold stress, although the response was delayed in the root (Figure 5). Similar results were obtained when all other abiotic stresses were included in the PCA (Supplementary).
Figure 6. Principal component analysis of the cold (blue) drought (green) and UV-B light (red) data sets. Principal component analysis was applied to 40 stress treatment samples exhibiting clear differences for specific stresses, tissues, and time points. The 40 stress samples included root (circles) and shoot (triangles) tissue for the indicated time points (see Experimental procedures).
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Global changes in gene expression in response to UV-B light
We next performed a more detailed investigation of spatio-temporal changes in the number of differentially expressed genes during the UV-B light-induced response. The UV-B light stress was of special interest because it produced a very fast and transient gene expression response and included a systemic shoot-to-root signalling aspect (Figure 2).
This analysis revealed that 100 genes in the shoot and 39 genes in the root displayed a strong upregulation 15 min after the onset of UV-B light irradiation (Figure 7). Fifteen minutes later this number increased to around 300 in the shoot and 200 in the root. This increase continued until a maximum was reached after 6 h in the shoot and 3 h in the root (Figure 2, Table 1). Afterwards we observed a sharp drop of differentially regulated genes in both tissues. A similar pattern was recognizable for genes downregulated by UV-B (Figure 2).
Figure 7. Expression profiles of genes immediately upregulated in the root and the shoot after 15 min in response to irradiation with a 15-min UV-B light pulse. Time course of the relative expression levels of the genes differentially regulated between 0.25 and 0.5 h after the onset of UV-B light irradiation in the shoot (a) and the root (b). The upper left diagram in (a) and (b) shows the complete set of genes, whilst the other diagrams display the divergent expression kinetics of these genes within 30 min. The red line shows the expression level of these genes in mock-treated control plants. The identity of the genes is provided in Supplementary Table S4.
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A high proportion (18%; representation in the genome 12.4%) of genes upregulated in shoots within 30 min of the onset of irradiation represent transcriptional regulators of various families. Among these we identified members of the B-box-, CCCH-, C2H2- and C3HC4-type zinc finger proteins, WRKY transcription factors, transcriptional regulators involved in the ethylene response pathway (ERFs) and those participating in auxin signalling (Supplementary). In addition, genes likely to encode signalling components, as for example different types of kinases and proteins involved in Ca2+ signal transduction, were identified among the immediate response genes (Supplementary). Notably, although the plants were grown under sterile conditions and had not been exposed to any pathogen, a large number of genes annotated to be involved in pathogen signal transduction and defence response as well as in oxidative stress signalling and adaptation were upregulated by irradiation with UV-B light (Supplementary). Whereas the proportion of genes coding for transcriptional regulators, signalling elements and pathogen-related proteins stayed high over the entire time period investigated, the genes representing components of certain metabolic pathways (e.g. flavonoid biosynthesis), elements of the chaperone/protein degradation machinery and various transporters were predominantly induced at later time points (1–6 h after onset of irradiation) in both roots and shoots (data not shown).
The general expression patterns presented in Figure 6 indicate that Arabidopsis responds to a 15-min pulse of UV-B light by a transient induction of thousands of genes. However, a detailed view of the kinetics revealed that this pattern comprised, on the one hand, expression transients occurring within a short period of minutes or a few hours and, on the other, sustained expression over a long period of several hours (Figures 7 and 8). For instance, of the 100 genes induced in the shoot after 15-min UV-B light, 26 were no longer upregulated 15 min later (Figure 7b). However, the transcripts of some of these genes reappeared a few hours later indicating a second transient of UV-B light-induced gene expression (Figure 8). A similar expression profile was observed in the root (data not shown). It is noteworthy that the major proportion (30.8%) of these 26 transiently expressed genes encode kinases, Aux/IAA proteins and, particularly, components involved in pathogen defence and, to a minor extent, transcriptional regulators (Supplementary). This transient expression principle was also observed when later time points were included in the analysis. Of the 535 genes upregulated in the shoot 1 h after UV-B light treatment only 67 were already induced at 15 min and 211 at 30 min, respectively (data not shown). Thus, a large proportion of UV-B light-regulated gene expression, which is very likely to be responsible for the initiation of biochemical, physiological and morphological adaptation responses, appears to happen in the first response phase of 30 min and in a second response phase several hours later.
Figure 8. Differential expression pattern of genes upregulated by a 15-min UV-B light pulse. Kinetic clusters of gene expression pattern in the shoot induced by a 15-min UV-B light pulse. All upregulated genes could be included in one of the kinetic clusters. The black lines show representative transcriptional regulators. Clustering was performed as described in Experimental procedures.
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Due to the design of the growth boxes, UV-B light was exclusively absorbed by the aerial parts of the irradiated plants. The concurrent harvest of shoots and roots from identically treated plants offered us the unique possibility of determining the extent to which UV-B irradiation regulates common or different genes in the shoot and the root – an approach which was not previously possible (Brown et al., 2005; Ulm et al., 2004). At early time points (15 to 60 min after the onset of irradiation) only around 10% of genes upregulated by UV-B light were identical in the shoot and the root tissue (Table 3, Supplementary). At later time points the proportion of identical genes declined even more. The low percentage of commonly expressed genes as well as the downward tendency over time was more pronounced in the case of UV-B downregulated genes (Table 3). In the initial phase (15 min after the onset of irradiation) around 10% of UV-B light-repressed genes were identical in both tissues. This proportion decreased continuously at later time points (Table 3).
The genes upregulated in common in the roots and the shoots within the first hour after UV-B light exposure encode transcriptional regulators of various families (19.8%), signalling elements and components participating in pathogen signal transduction or defence (Supplementary). The upregulation of long hypocotyl in far-red (HFR1) and long hypocotyl 5 (HY5) suggests that components of the UV-B stress response pathway and elements required for the photomorphogenic UV-B light response pathway (Ulm and Nagy, 2005) were induced in both roots and shoots. Furthermore, we observed a fast and transient upregulation of the PHR1 gene, of which the gene product, a CPD photolyase, plays a major role in the repair of UV-B light-induced DNA damage.
The overall overlap of our UV-B light stress data with the data set published by Ulm et al. (2004) is shown in Supplementary. Whereas Ulm et al. observed 660 (1716) genes differentially regulated 1 h (6 h) after onset of the irradiation, 758 (1274) genes responded in this study. There was overlap of 383 (1 h) and 816 (6 h) genes found in both studies, unravelling a robust set of UV-B light-inducible genes, although – with the exception of the UV-B light source and intensity used – the experimental set-up was different. Furthermore, with one exception, the 39 genes responsive to UV-B light found by Brown et al. (2005) to be transcriptionally dependent on the activity of UVR8 and HY5 were also found in our data set (Supplementary). However, the vast majority of the genes differentially regulated by damaging UV-B light under our conditions were not identified by Brown et al. (2005) suggesting that they are transcriptionally independent of the UVR8 and HY5 gene products.