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Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 RESEARCH ARTICLE Open Access Seed-specific elevation of non-symbiotic hemoglobin AtHb1: beneficial effects and underlying molecular networks in Arabidopsis thaliana Johannes Thiel1, Hardy Rolletschek1*, Svetlana Friedel1, John E Lunn2, Thuy H Nguyen3, Regina Feil2, Henning Tschiersch1, Martin Müller1, Ljudmilla Borisjuk1 Abstract Background: Seed metabolism is dynamically adjusted to oxygen availability. Processes underlying this autoregulatory mechanism control the metabolic efficiency under changing environmental conditions/stress and thus, are of relevance for biotechnology. Non-symbiotic hemoglobins have been shown to be involved in scavenging of nitric oxide (NO) molecules, which play a key role in oxygen sensing/balancing in plants and animals. Steady state levels of NO are suggested to act as an integrator of energy and carbon metabolism and subsequently, influence energy-demanding growth processes in plants. Results: We aimed to manipulate oxygen stress perception in Arabidopsis seeds by overexpression of the nonsymbiotic hemoglobin AtHb1 under the control of the seed-specific LeB4 promoter. Seeds of transgenic AtHb1 plants did not accumulate NO under transient hypoxic stress treatment, showed higher respiratory activity and energy status compared to the wild type. Global transcript profiling of seeds/siliques from wild type and transgenic plants under transient hypoxic and standard conditions using Affymetrix ATH1 chips revealed a rearrangement of transcriptional networks by AtHb1 overexpression under non-stress conditions, which included the induction of transcripts related to ABA synthesis and signaling, receptor-like kinase- and MAP kinase-mediated signaling pathways, WRKY transcription factors and ROS metabolism. Overexpression of AtHb1 shifted seed metabolism to an energy-saving mode with the most prominent alterations occurring in cell wall metabolism. In combination with metabolite and physiological measurements, these data demonstrate that AtHb1 overexpression improves oxidative stress tolerance compared to the wild type where a strong transcriptional and metabolic reconfiguration was observed in the hypoxic response. Conclusions: AtHb1 overexpression mediates a pre-adaptation to hypoxic stress. Under transient stress conditions transgenic seeds were able to keep low levels of endogenous NO and to maintain a high energy status, in contrast to wild type. Higher weight of mature transgenic seeds demonstrated the beneficial effects of seed-specific overexpression of AtHb1. Background Hemoglobins (Hbs) represent a large ubiquitous group of proteins found in all kingdoms of life [1]. In plants, there are three major groups: (i) symbiotic or leghemoglobins, facilitating oxygen diffusion to nitrogen-fixing bacteria in nodules of plants (ii) non-symbiotic hemoglobins (nsHbs) found in numerous species, and (iii) the * Correspondence: rollet@ipk-gatersleben.de 1 Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, 06466 Gatersleben, Germany Full list of author information is available at the end of the article poorly characterized group of truncated hemoglobins [2,3]. The nsHbs in turn are divided into class-1 (Hb1) and class-2 (Hb2) subgroups based on phylogenetic analyses and structural/kinetic properties of the proteins. Hb1 has a superior affinity for oxygen and its expression is induced during hypoxic stress [4,5]. Notably, its overexpression in plants was shown to enable the cell to maintain high ATP levels under hypoxia [6]. This finding was later explained by the ability of Hb1 to detoxify reactive nitrogen species like nitric oxide (NO) [7,8]. NO is a key signaling molecule involved in multiple © 2011 Thiel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 processes, like stomatal closure, programmed cell death and pathogen resistance [9]. The level of NO rises under hypoxia, and is related to the availability of nitrite [4,5,10]. Despite the clear effects of Hb1 on the abundance of NO, the in vivo sources of NO, its targets as well as signaling mechanisms are still a matter of debate [11]. Seeds of crop species experience a regular oxygen deficiency during both development and germination [12]. This leads to ATP limitation and subsequently, to a restriction of high energy-demanding processes like cell division, growth and storage product synthesis [13]. Oxygen limitation is in part caused by the high diffusional impedance of certain seed structures. Thus, even the tiny seeds of Arabidopsis thaliana operate close to the edge of hypoxia. Consequently, a moderate decrease in atmospheric oxygen concentration to about half saturation already induces clear metabolic restrictions in Arabidopsis seeds [14]. The molecular mechanisms of the seeds’ response to hypoxia might resemble those of other plant organs [15-17] and tissue types [18] of Arabidopsis, but detailed transcriptomic studies are lacking. Based on a series of in vitro experiments, we recently proposed that the steady state level of NO in seeds acts to integrate carbon and energy metabolism [5]. Upon application of either NO scavengers or NO inducing compounds, seeds responded with alterations in both oxygen uptake and metabolic activity evident at both the transcript and metabolite level. Congruently, respiratory activity of isolated seed mitochondria showed clear responses to NO/nitrite [10]. However, the extent to which such in vitro studies mirror the in vivo situation can always be questioned. Here, we used the non-symbiotic hemoglobin AtHb1 to manipulate endogenous levels of NO in seeds. The AtHb1 (also referred to as AtGLB1 or AHb1 in the literature) was overexpressed under the control of the seed-specific LeB4 promoter in Arabidopsis thaliana. Comparative analyses of both transcripts and metabolites were performed with wild type (WT) and transgenic plants grown under standard conditions as well as under moderate hypoxic stress treatment. Results indicate that AtHb1 overexpression led to several alterations in transcriptional and metabolic networks, resulting in improved seed yield (weight). Results Overexpression of AtHb1 is targeted to seed and increases seed weight We generated transgenic Arabidopsis plants expressing the endogenous AtHb1 under the control of the seedspecific LeB4 promoter [19]. Northern blot analysis of siliques from homozygous T3 plants demonstrated significant AtHb1 expression, whereas in WT plants the Page 2 of 18 endogenous AtHb1 expression was not detectable under standard conditions (Figure 1A; for additional transgenic lines see below). RT-PCR analysis showed that, overexpression of AtHb1 under the control of the LeB4 promoter was restricted to siliques/seeds in the transgenic plants (minor expression in roots; Figure 1B). Comparison of manually isolated seeds with whole siliques (including seeds) revealed that LeB4-driven expression is mainly localized in seeds in agreement with previous results [19]. To avoid any stress-induced artefacts that might be induced by dissection of seeds from the siliques, whole siliques were used for further studies AtHb1 overexpression did not alter the vegetative growth of transgenic plants. Also timing of developmental programmes, like induction of flowering and silique development were not affected by transgene expression. Interestingly, mature seeds of transgenic plants revealed a higher weight (Table 1) whereas seed number and composition were unaffected. Overexpression of AtHb1 reduces the endogenous level of nitric oxide in seeds A qualitative fluorescence assay with diaminofluoresceine-2-diacetate (DAF-2DA) was used for detection of endogenous NO in WT and AtHb1 embryos under standard and hypoxic stress conditions. To induce moderate hypoxic stress in the seeds, intact plants were treated with artificial air mixes containing only 10.5 kPa oxygen (corresponding to half atmospheric oxygen saturation) for one hour. Seeds of WT plants showed a slight induction of AtHb1 expression under these conditions (Figure 1C), but its expression level was still much lower than in the transgenic plants. Microarray results confirmed the higher abundance of AtHb1 mRNA in transgenics under hypoxia (>3-fold, Figure 2A, marked by asterisk). Under standard growth conditions, NO was not detectable in the embryos of either WT or AtHb1 plants using the fluorescence assay. Possibly, the steady state level of NO was below the detection limit of the assay. Under moderate hypoxia, WT showed a clear fluorescence signal (in green), while AtHb1 overexpressors did not (Figure 1D). This indicated strongly decreased NO levels in the latter. Thus, the transgenic approach resulted in lower levels of NO. The induction of AtHb1 expression (Figure 1C) and enhanced NO emission (Figure 1D) in WT further indicated that the moderate stress treatment was sufficient to induce hypoxia in seeds. Experimental set up for microarray analysis To assess changes in gene expression in seeds/siliques due to AtHb1 overexpression in detail, we focused on line L1-1, which showed the strongest transgene Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Page 3 of 18 Figure 1 Effects of AtHb1 overexpression in Arabidopsis seeds. (A) Northern blot analysis of AtHb1 expression in WT and homozygous transgenic plants (L1-1 and L1-4) at 45 DAG, 25S RNA was used as loading control. (B) RT-PCR analysis of AtHb1 expression in different tissues of L1-1. (C) RT-PCR analysis of AtHb1 expression in siliques of WT and L1-1 under control conditions and moderate hypoxia. (D) Fluorescence detection assay of NO using DAF-2DA. Fluorescence signals (green) indicate NO accumulation. expression. Six other independent transgenic lines were involved in further studies (see below). WT and transgenic plants were exposed to moderate hypoxia (10.5 kPa) or normoxia (21 kPa; control) for one hour. Three biological replicates were used for hybridization to Affymetrix ATH1 arrays. A cluster dendrogram of transcript signal intensities from the 12 arrays showed a high reproducibility of the biological replicates from each data set (genotype+treatment), and indicated a greater influence of the genotype than the treatment on transcriptional profiles (Additional file 1A). Transcript analysis by qRT-PCR showed a high correlation (R2 = 0.83) with the microarray data, confirming the reliability of the data (Additional file 1B). We compared the transcriptome of WT and AtHb1 siliques/seeds under control and hypoxic conditions, as well as the hypoxic responses in each genotype. Differentially expressed genes were extracted from the data base by applying the following cutoffs: a fold-change of Table 1 Characteristics of mature seeds of WT and AtHb1overexpressing lines WT Line 1-1 Line 1-4 Total lipid (% DW) 34.8 ± 3.0 36.2 ± 6.5 29.4 ± 10.2 Total protein1 (% DW) 22.6 ± 2.0 21.4 ± 0.6 23.0 ± 1.3 Total carbon (% DW) 53.1 ± 1.4 54.9 ± 1.4 53.7 ± 1.2 Seed weight2 (μg) 17.8 ± 3.5 23.0 ± 3.2 21.1 ± 2.3 % increase in seed weight 100 131 ± 18 130 ± 15 13231 ± 2576 16851 ± 4685 15115 ± 2273 Seed number per plant3 Data are means (± SD). Bold values indicate statistically significant differences (t-test, p < 0.05). 1 calculated from total N content * 6.25 2 analysed in three generations (T3-T5) 3 calculated from seeds per pod * pods per plant >2 and a p-value of <0.05. A total of 1,010 genes were identified as differentially expressed in all of the comparisons. Differentially expressed genes were grouped into eight clusters (Additional file 2 and 3), classified into functional groups using the MapMan bin code [20] and ordered by pathways. The heat map display in Figure 2 gives a detailed view of the altered pathways (also listed in Additional file 4). To confirm that microarray data of L1-1 are reproducible in further transgenic lines, we analyzed the expression of selected genes in six other AtHb1-overexpressing lines by qRT-PCR (Figure 3). A set of transcripts that have been shown in the microarray analysis to be upregulated by AtHb1 overexpression was selected for qRTPCR analysis. All of the transgenic lines exhibited an enhanced expression of the genes from representative signaling, redox and metabolic pathways compared to the WT, indicating similar expression profiles due to AtHb1 overexpression in independent transgenic lines. AtHb1 overexpression induces stress-related regulatory pathways under non-stress conditions Comparison of the transcriptome of WT and AtHb1 overexpressors under control conditions revealed multiple changes (Table 2). The effects on molecular networks involved in stress responses and signaling were particularly pronounced (Figure 2A). WRKY and AP2/EREBP transcription factors, as well as genes related to hormone metabolism, i.e. abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA), were found to be upregulated in AtHb1 seeds. Moreover, many genes involved in signaling processes, like MAPK kinases and receptor kinases, and in redox/stress-related processes were strongly induced. This trend was also confirmed by analysis of differentially expressed genes for indicative over- and underrepresented gene ontology categories (GO terms). Upregulated Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Page 4 of 18 Figure 2 Heat map display of differentially expressed genes involved in regulation/redox processes and primary metabolism. Columns indicate mean signal log2 ratios of differentially expressed genes in at least one comparison. Each comparison is arranged into vertical columns in the following order: column 1, AtHb1 overexpression versus WT under control conditions; column 2, comparison of both genotypes under hypoxic conditions; column 3, WT under hypoxia versus WT under control conditions; column 4, AtHb1 under hypoxia versus AtHb1 under control conditions. Blue indicates downregulation, yellow indicates upregulation. Genes organized by pathways, (A) regulation/signaling and stress response, (B) primary metabolism and transport. Additional file 4 contains the gene lists used. genes in AtHb1-overexpressing plants showed a strong enrichment of GO categories involved in stress responses (Additional file 5). Among transcription factors, four transcripts, encoding WRKY 33, 40, 53 and 75, were significantly upregulated. WRKY genes have been shown to play a role in hypoxic responses of different cell types of Arabidopsis [18]. Prominent differences in hormone metabolism were observed for ABA, SA and auxin-related genes. A strong upregulation of NCED4 was accompanied by preferential expression of transcripts encoding ABAresponsive proteins (At2g40170, At3g02480, At5g62490). Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Page 5 of 18 L1_4/WT 4.5 WT L1_4 L2_3 L2_9 L2_11 L2_15 L2_16 L2_3/WT L2_9/WT AtHb1 L2_11/WT 3.5 L2_15/WT ǻǻCt L2_16/WT 2.5 1.5 0.5 ICL MS GS ATPase MnSOD -0.5 AOX1 TPS8 MAPKK9 WRKY 53 WAK1 genes Figure 3 Transcript ratios of AtHb1-induced marker genes in different AtHb1-overexpressing lines relative to WT. AtHb1 transcript accumulation in siliques of different transgenic lines obtained by RT-PCR is depicted in the inset. For transcript analysis siliques of 45 DAG plants have been used. qRT-PCR analysis was conducted for genes showing a preferential expression in AtHb1 (Line 1-1) compared to WT under control conditions as measured by microarray analysis. MnSOD (At3g56350), ICL (At3g21720), MS (At5g03860), AOX1 (At1g32350), WAK1 (At1g21250), GS (At5g53460.), ATPase (Chl) (At1g15700 ), TPS8 (At1g70290), MAPKK9 (At1g73500), WRKY 53 (At4g23810). The elevation of transcripts involved in ABA metabolism/signaling is consistent with an overrepresentation of ABRE binding sites in the 5’-flanking regions of AtHb1 coexpressed genes (Table 3). Auxin transport and signaling is commonly downregulated in transgenics. Fourteen genes, among them auxin transporter (AUX1), auxin-induced genes (GH3, SAUR, IAA, ARF1) were strongly downregulated, whereas two transcripts encoding auxin downregulated protein ARG10 were upregulated. Genes implicated in signaling pathways, like receptor kinases, wall-associated kinase 1 (WAK1, At1g21250) and MAPK kinase 9 (At1g73500) were also upregulated compared to WT. WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain bound to the pectin fraction of cell walls [21], thus enabling communication between cell wall and cytoplasm. Phosphorylation via WAKs has been shown to play a pivotal role in cell wall metabolism [22], which was significantly altered by AtHb1 overexpression. WAK1 expression is induced by SA treatment [23], thus, higher expression of WAK1 and two S-adenosyl-L-methionine:carboxyl methyltransferases indicates an involvement of SA signaling in the regulatory networks controlled by AtHb1. In addition, the expression of 11 transcripts encoding receptor kinases, such as transmembrane kinase RLK5 and other leucine-rich repeat family proteins as well as Ser/Thr kinases, revealed the presence of different signaling pathways. Interestingly, RLK7 (At1g09970) has recently been shown to be involved in the control of seed germination and tolerance to oxidative stress [24]. Using Table 2 Number of differentially expressed genes Number of genes AtHb1_control vs. WT_control AtHb1_hyp vs. WT_hyp WT_hyp vs. WT_control AtHb1_hyp vs. AHb1_control upregulated 270 176 351 153 downregulated 205 197 62 101 Genes with log2 signal ratios > 1 and p-values < 0.05 between WT and AtHb1-overexpressing plants under control and hypoxic conditions and after hypoxic treatment of each genotype were extracted from the data base. Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Page 6 of 18 Table 3 Promoter motifs of differentially expressed genes Motif (1000 bp upstream) p-value AtHb1 vs. WT upregulated control Motif (1000 bp upstream) p-value AtHb1 vs. WT downregulated control ABRE-like binding site motif < 10e-10 MYCATERD1 < 10e-5 ABRE binding site motif < 10e-5 AtMYC2 BS in RD22 < 10e-5 ACGT ABRE motif A2OSEM < 10e-10 ABREATRD22 < 10e-5 GADOWNAT < 10e-10 Ibox promoter motif < 10e-5 Z-box promoter motif CACGTG motif < 10e-10 < 10e-10 AtHb1 vs WT upregulated hypoxia AtHb1 vs WT downregulated hypoxia no enrichment WT hyp vs WT control upregulated < 10e-7 < 10e-7 RY-repeat promoter motif < 10e-6 WT hyp vs WT control downregulated W-box/WRKY < 10e-5 I-Box ABRE-like binding site motif < 10e-7 < 10e-9 ABRE binding site motif < 10e-7 ACGT ABRE motif A2OSEM < 10e-10 DRE core motif < 10e-8 DREB1A/CBF3 < 10e-6 CACGTG motif < 10e-10 GADOWNAT < 10e-10 AtMYC2 BS in RD22 MYCATERD1 < 10e-5 < 10e-5 Z-box promoter motif < 10e-7 EveningElement promoter motif < 10e-5 AtHb1 hyp vs AtHb1 control upregulated EveningElement promoter motif MYCATERD1 AtMYC2 BS in RD22 no enrichment AtHb1 hyp vs AtHb1 control downregulated < 10e-5 ABRE-like binding site motif < 10e-7 ABRE binding site motif < 10e-5 ACGT ABRE motif A2OSEM < 10e-9 G-box LERBC GADOWNAT < 10e-5 < 10e-9 RY-repeat promoter motif < 10e-6 Overrepresented motifs with p-values < 10e-4 were selected for comparative analysis. genetic approaches the authors provided evidence for a positive correlation of RLK7 expression and enhanced tolerance against H2O2. Transcripts encoding proteins involved in redox homeostasis, such as manganese superoxide dismutase (MnSOD, At3g56350) and two glutathione-S-transferases, were upregulated in AtHb1 overexpressors. This was accompanied by higher expression of defencerelated proteins, i.e. dehydrins and major latex proteins (MLP-related) (Figure 2A). Ubiquitin-mediated proteolysis is essential for plant development and responses to environmental stimuli [25]. AtHb1 induced the expression of three RING finger E3 ligases of the C3CH4-type (At4g14365, At2g27940, At1g30860) and two F-box proteins (SKP1/ At2g45950 and kelch repeat/At1g80440) (Additional file 6). RING finger ligases and E3 ligases from the SKp1, Fbox (SCF) complex play an essential role in auxin metabolism by degrading AUX/IAA proteins, and thereby regulating concentrations of IAA [25]. This is probably linked to downregulation of auxin transport and signaling in AtHb1 plants. AtHb1 overexpression in seeds alters expression of genes involved in primary metabolism AtHb1 overexpression induces various changes in transcripts related to carbohydrate, cell wall, N- and lipid metabolism, as well as potentially associated transporter gene activities and photosynthesis. As deduced from GO analysis of transcript data, the cell wall was the most Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 affected cellular compartment in AtHb1 seeds showing a clear underrepresentation (Additional file 5). Other decreased biological processes are linked to cell wall biogenesis and modification. This is illustrated by the concurrent downregulation of more than 30 cell wallrelated genes encoding cellulose synthases, arabinogalactan-proteins (AGPs), pectinesterases, expansins, xyloglucan-xyloglucosyl transferases and polygalacturonases (see MapMan visualization, Additional file 7). This indicates a strong repression of cell wall synthesis, cell wall modification, pectin degradation, cell expansion and cell wall turnover. Two transcripts (At1g70290, At2g18700) encoding class II trehalose-6-P synthase/phosphatase (TPS8, TPS11) were preferentially expressed in AtHb1 plants. These transcripts are also potentially linked to cell wall metabolism, as it was found that perturbation of trehalose metabolism in embryos of the tps1 mutant leads to changes in cell wall composition and thickness [26]. Lipid metabolism also showed transcriptional alterations; fatty acid elongation and desaturation were activated but transcripts involved in squalene and steroid metabolism were repressed. In addition, transcripts for malate synthase and isocitrate lyase (key enzymes of the glyoxylate pathway) were upregulated in AtHb1 seeds. Furthermore, transcripts encoding the 4Fe-4S cluster protein of photosystem I and key enzymes of the photorespiratory pathway (glycolate oxidase/GOX, At3g14415; serine hydroxymethyltransferase 4/SHMT4, At4g13890) were downregulated. Nitrogen metabolism appears to be affected in AtHb1 seeds based on the downregulation of nitrate reductase 2 (NIA2, At1g37130) and nitrite reductase 1 (NiR1, At2g15620). Several transcripts involved in amino acid metabolism differed significantly between transgenic and WT (S-adenosylmethionine synthetase, S-adenosyl-Lhomocysteinase, asparaginase, cystine lyase, delta-1-pyrroline-5-carboxylate synthetase). Several transporter gene activities were commonly downregulated in AtHb1 seeds, namely those involved in sugar, amino acid and oligopeptide transport (POT). Most of these are proton-coupled transporters. In addition, five genes from different subgroups of the aquaporin family were downregulated. These genes play a role in nutrient flow and/or are implicated in remobilization [27,28]. Changed gene interactions due to AtHb1 overexpression point to alterations in cell wall metabolism To infer gene-to-gene interactions we used the MRNET approach which extracts statistical dependencies between genes [29]. The reconstructed network of gene interference for the top 20 genes that are differentially expressed between WT and AtHb1 overexpressing seeds under control conditions showed clear differences (Additional Page 7 of 18 file 8). In WT, the gene encoding fasciclin-like arabinogalactan protein 13 (FLA13; At5g44130) was the central hub. AGPs, such as FLA13, play a role in plant cell elongation/cell wall biogenesis, and are assumed to act as signal molecules [30]. Proteins containing fasciclin domains have also been shown to function as adhesion molecules in a broad spectrum of organisms [31]. There were multiple interactions of this hub with genes encoding proteins localized to the cell wall (e.g. xyloglucan:xyloglucosyl transferase, xyloglucan endotransglycosylase 3 (XTR3), proline-rich protein 2 (ATPRP2) and acid phosphatase class B family protein) or otherwise involved in extracellular matrix modifications (e.g. midchain alkane hydroxylase, which is involved in cuticular wax biosynthesis; [32]). Most of the genes are implicated in stressresponses and related to hormone (ABA, GA) action. Overexpression of AtHb1 directly or indirectly perturbed the strong multiple interactions of the hub gene FLA13, shifting the main regulatory point to ATPRP2. It has been shown, that ATPRP2 is one of the key genes involved in cell specification [33]. Cell specification in the embryo might be coupled to maturation processes, which are characterized by high storage- but extremely low mitotic-activity. Downregulated expression of ATPRP2 (and associated genes) in AtHb1 plants might therefore indicate decelerated cell specification and thus, an extented growth phase. Evaluation of adaptive stress responses in wild type seeds Most of the adaptive responses in WT seeds have also been described for shoots and roots of Arabidopsis plants. Mustroph et al. [18] identified a core set of 49 translated hypoxia-induced mRNAs in 21 different Arabidopsis cell populations. From this core set, 35 genes (~70%) were also found to be upregulated in seeds, indicating similar adaptation strategies to hypoxia regardless of tissue/organ identity. The possible induction of the glyoxylate cycle in combination with lipid degradation (phospholipase C, phosphodiesterase) was not observed in other Arabidopsis tissues and might therefore be seed-specific. The induction of the glyoxylate cycle could represent an alternative mechanism to generate sugars and sustain energy supply under unfavourable conditions in seeds. Interestingly, malate synthase and isocitrate lyase are also enhanced in carbon-starved cucumber cotyledons [34]. The higher expression of genes involved in sugar, amino acid, oligopeptide and general nutrient (aquaporins) transport in WT (column 2 in Figure 2B) and the significantly reduced sucrose concentrations (see below) indicates nutrient, particularly sugar, depletion in WT upon hypoxia. In general, WT seeds showed a strong transcriptional and metabolic response to moderate hypoxia. Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Metabolism and signaling of hormones (ABA, ethylene, JA, SA and GA) which are described to be important triggers in response to oxidative stress [15,16] are strongly induced in seeds. Activation of specific transcription factors and signaling pathways nicely illustrates a cross-talk of hormone action and regulatory pathways, particular for ethylene. Upregulation of MAPKK9, MAPK3 (At3g45640) accompanied by activation of ACC oxidase1 (At2g19590) as well as ten members of the AP2/EREBP family represents an example how signaling cascades are linked together in adaptive stress responses. Experiments with maize suspension cultures showed a correlation of varying class-1 hemoglobin levels and changed NO concentrations with ethylene formation [35]. Enhanced ethylene biosynthesis under hypoxia is linked to lower hemoglobin expression, coinciding with the stronger induction of ethylene synthesis and signaling in the WT compared to the AtHb1 plants in our experiments. Beside the strong activation of several WRKY transcription factors and MYB44 (At5g67300), transcripts related to redox regulation were clearly induced. Rising concentrations of H2O2 in WT upon hypoxia correlate with transcriptional activation of several ROS generating/scavenging enzymes coinciding with other studies [36,37]. The upregulation of several class II TPS genes and the reduction of trehalose-6-P (T6P) levels was part of the hypoxic response in WT (two of them are also induced in transgenics under control conditions). Interestingly, T6P metabolism was identified as being part of a hypoxic response that is conserved in some pro- and eukaryotes [38]. T6P may be involved in coordination of carbon partitioning between primary metabolism and cell wall synthesis [39]. Therefore, altered expression of TPS genes together with changes in cell wall metabolism - accentuates the possible role of T6P metabolism in regulation of carbon partitioning. In general, the alterations in regulatory and metabolite pathways provide a framework of seed-specific responses to hypoxia. AtHb1 overexpression attenuates transcriptional stress responses Under hypoxic stress treatment, a significantly lower number of transcripts exhibited altered expression in AtHb1 compared to WT (254 and 413 genes, respectively). Consequently, the stress response observed in AtHb1 was much reduced, especially in regulatory/signaling pathways, but also for specific pathways in primary metabolism. Transcriptional alterations in WT upon hypoxia partly shared a commonality with those induced by AtHb1 overexpression under control conditions, or with transcripts additionally induced in AtHb1overexpressing plants after hypoxia (Figure 4). The moderate hypoxic response in seeds of transgenic plants, in Page 8 of 18 Enhanced AtHb1/WTnormox AtHb1-hypoxia 153 0 52 154 272 16 85 102 148 WT-hypoxia 351 Repressed AtHb1/WTnormox AtHb1-hypoxia 101 0 97 181 205 0 24 4 34 WT-hypoxia 62 Figure 4 Venn diagrams showing overlap of differentially expressed genes due to AtHb1 overexpression and genes involved in the hypoxic response of WT and/or AtHb1 plants. Overlap of differentially expressed genes was identified using the Venn Super Selector of the web-based tool BAR (http://bbc.botany. utoronto.ca/). combination with genes induced by AtHb1 overexpression that have been shown to be implicated in the WT hypoxia response, points to a kind of “pre-adaptation” to oxidative stress. Among the differences between the two genotypes in their hypoxic responses, several biological processes stand out, namely, stress-related signaling, redox pathways and primary/energy metabolism (Figure 2, Additional file 4). These differences are discussed in detail below. First, hypoxia induced stress-related signaling and redox pathways in WT. GO analysis for functional assignments of upregulated genes showed strong overrepresentation of responses to abiotic/biotic stress and other biological processes related to stress responses, especially responses to ABA and JA. Evaluation of promoter motifs within the 5’-flanking regions of hypoxiainduced genes revealed that W-box, ABRE, DREB, Gbox, MYC2, MYCATERD1, GADOWNAT, Z-box, I-box and Evening Element motifs were significantly overrepresented. This finding is significant because almost all Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 of these recognition sites have been implicated in hormone signaling (ABA, ethylene) and in general stress responses. In addition to these changes in hormone signaling pathways, transcripts directly involved in biosynthesis of ABA, ethylene, JA and SA were commonly upregulated in WT. In contrast genes related to SA, GA and ABA metabolism were not induced by hypoxia in AtHb1 plants. In fact, a strong repression of ABA synthesis/signaling was evident from the down regulation of NCED4 and several ABA-responsive genes, among them ATEM6 and AtHVA22b (which were already induced under control conditions by AtHb1 overexpression). In addition, ABRE binding site motifs were enriched in the set of downregulated genes in AtHb1 plants after hypoxia (Table 3). Another striking difference between the genotypes is the opposite regulation of transcripts encoding the gibberellin regulated proteins 2 and 3 (GASA 2, 3); they are highly upregulated in the WT after hypoxic treatment whereas a strong repression was observed in transgenic seeds. Calcium signaling seems to play a role in the hypoxic response of WT, as indicated by the upregulation of six transcripts encoding calmodulins and calmodulin binding proteins, accompanied by an induction of calcium dependent protein kinase and the plastidic Ca 2+ -ATPase1 (ACA1, At1g27770). The transcriptional activation of calmodulins which are the primary calcium receptors in plant cells and calcium binding proteins, could serve as substrate for phosphorylation by calcium dependent protein kinases, then activating transcription factors by phosphorylation. Altogether this points to existing calcium dependent signaling pathways in the hypoxia response in wild type seeds, which were not observed in AtHb1 overexpressors. The second major difference between AtHb1-overexpressing plants and WT concerned primary and energy metabolism. Hypoxia induced multiple changes in transcripts related to these processes in WT, but only moderate changes in AtHb1 plants. For example, in WT we encountered a clear induction of glycolysis and fermentation (FBP aldolase, PFK, PDC1, ADH1) as well as strongly induced nitrogen assimilation as suggested by preferential expression of NIA2 and NiR1. In WT, cell wall metabolism was downregulated as evidenced by repression of six transcripts encoding pectinesterases and four encoding polygalacturonases, indicating that cell wall metabolism is one of the key processes affected by hypoxia. Induction of carbonic anhydrases and genes implicated in lipid degradation and the glyoxylate cycle (malate synthase, isocitrate lyase) was apparent in the WT response but not in AtHb1 plants. The activity of transporter genes is directly linked to primary metabolism. The strong induction of genes encoding proline transporter, POT as well as TIP1.2 and TIP3.2 is also Page 9 of 18 restricted to the hypoxia response in WT and might reflect a higher demand for remobilizing storage compounds and thus, indicating nutrient depletion in WT. The alterations observed in the transgenic plants were restricted to upregulation of glycolysis/fermentation (PFK, PDC1, ADH1) and a few transcripts related to cell wall degradation. AtHb1 plants show less pronounced metabolic adjustment under transient hypoxia The steady state level of amino acids, sugars, metabolic intermediates and H2O2 were measured in seeds/siliques of both genotypes under control and hypoxic conditions. Under control conditions, the levels of phosphoglycerate and ADP-glucose (starch precursor) were higher in WT versus AtHb1 plants, while sucrose and UDP-glucose (cell wall precursor), showed elevated levels in AtHb1 plants (Figure 5, values are given in Additional file 9). Remarkably, the levels of many metabolites changed after hypoxic treatment in WT but were barely altered in AtHb1 plants. In WT plants only, the levels of T6P and sucrose dropped significantly, while pyruvate increased (indicative of enhanced glycolytic flux and/or a partial block of the TCA cycle). Altogether, the metabolite profiles of the two genotypes illustrated a strong metabolic adjustment in WT in response to moderate hypoxia, whereas in AtHb1 only marginal changes were detected. This differential response was clearly visualized using principal component analysis (PCA; insert in Figure 5). Transcript data hinted at shifts in ROS metabolism in transgenic plants and in the hypoxic response of WT. Measurements of H2O2 levels in both genotypes under control and hypoxic conditions are consistent with transcriptional activities of H2O2 generating and scavenging enzymes. Higher concentrations in AtHb1 seeds/siliques compared to WT under control conditions (Figure 6A) correlate with preferential expression of MnSOD1 and glutathione-S-transferases. Upon hypoxia, H2O2 levels in WT increased but were unchanged in AtHb1 seeds. Activation of respiratory burst oxidase homologue D, MnSOD1, redoxins, three glutathionine-S-transferases and alternative oxidase 1D (AOX1D, At1g32350) in WT indicates an enhanced ROS metabolism under hypoxia. Overexpression of AtHb1 promotes respiration and maintains the energy status under transient hypoxia To investigate changes in energy metabolism we measured the respiratory activity of developing seeds. Under control conditions respiration rates were similar in both genotypes (1.7 ± 0.2 pmol/µg embryo min). However, under hypoxia, respiration in AtHb1 plants (line 1-1, 1.05 ± 0.14 pmol/µg min) was about 40% higher than in WT (0.73 ± 0.13 pmol/µg min) pointing to a higher Thiel et al. BMC Plant Biology 2011, 11:48 http://www.biomedcentral.com/1471-2229/11/48 Page 10 of 18 S6P a,b b * * a * a * a * b * b * b * a * b a * a * * a * a,b * a * a * b * b * a * a * b * b a a * * * a * b * a * a b * * a * b a * * b b * * a,b * a * b * b * b b * * a,b * a,b * a *b * a * a b * * b * a * a,b a * * *b a,b b * a * * b * a * b * b * a * a * b * a * a,b * b a,b * * b * *b a * a,b * a * b * a,b * b * Figure 5 Metabolite patterns in seeds of AtHb1-overexpressing and WT plants under control conditions (21 kPa O2) and moderate hypoxia (10.5 kPa O2) visualized by VANTED software [75]. “*a” indicates statistically significant differences after hypoxic treatment in each genotype, “*b” indicates statistically significant differences between the genotypes under control and hypoxic conditions (t-test, p < 0.05). Mean values ± standard deviation are presented (data in Additional file 9). The insert shows results of a principal component analysis of the metabolite data set. 20 samples in two dimensional space are given, where the names are coloured according to the 4 different sample types (WT and AtHb1, under either control or hypoxic conditions; with 5 biological replicates each).