A genome-wide analysis of the effects of sucrose on gene expression in Arabidopsis seedlings under anoxia

Abstract

Exogenous sucrose (Suc) greatly enhances anoxia tolerance of Arabidopsis (Arabidopsis thaliana) seedlings. We used the Affymetrix ATH1 GeneChip containing more than 22,500 probe sets to explore the anaerobic transcriptome of Arabidopsis seedlings kept under anoxia for 6 h in presence or absence of exogenous Suc. Functional clustering was performed using the MapMan software. Besides the expected induction of genes encoding enzymes involved in Suc metabolism and alcoholic fermentation, a large number of genes not related to these pathways were affected by anoxia. Addition of exogenous Suc mitigated the effects of anoxia on auxin responsive genes that are repressed under oxygen deprivation. Anoxia-induced Suc synthases showed a lower induction in presence of exogenous Suc, suggesting that induction of these genes might be related to an anoxia-dependent sugar starvation. Anoxic induction of genes coding for heat shock proteins was much stronger in presence of exogenous Suc. Interestingly, a short heat treatment enhanced anoxia tolerance, suggesting that heat shock proteins may play a role in survival to low oxygen. These results provide insight into the effects of Suc on the anoxic transcriptome and provide a list of candidate genes that enhance anoxia tolerance of Suc-treated seedlings.

Figure 1.

Pattern of mRNA accumulation for genes encoding known anaerobic proteins in Arabidopsis seedlings. A, Arabidopsis seeds were germinated under aerobic conditions for 4 d in liquid culture medium. Seedlings were kept under aerobic conditions (Air) or transferred to anoxia (Anoxia) for 1 to 72 h. B, Arabidopsis seeds were germinated under aerobic conditions for 4 d and subsequently transferred to anoxia for 10 to 60 min. RNA was extracted, electrophoresed, and northern analysis carried out using gene specific probes. Equal loading was checked by reprobing with a rRNA probe (not shown). A representative experiment is shown.

Figure 2.

Effects of exogenous Suc on seedling survival and mRNA accumulation. A, Arabidopsis seeds were germinated under aerobic conditions for 4 d on vertical agar plates containing Murashige and Skoog medium (0.5×) in presence or absence of exogenous Suc (90 mm). When added, Suc was present throughout the experiment. Plates were subsequently transferred to anoxia for 1 to 3 d, and recovery of seedlings was observed after 2 additional weeks of aerobic growth in the light. B, Effect of exogenous Suc added during the anoxic treatment on the expression of ADH and PDC1 genes. Arabidopsis seeds were germinated under aerobic conditions for 4 d in liquid culture medium and subsequently transferred to anoxia for 1 to 72 h. RNA was extracted, electrophoresed, and northern analysis carried out using gene-specific probes. The section reporting the results from the seedlings not treated with Suc was duplicated from Figure 1 to allow an easy comparison of the results. Equal loading was checked by reprobing with an rRNA probe (not shown). The quantitation of the mRNA in the northern analysis was performed and is reported in the graph above the respective northern-blot photograph. A representative experiment is shown. C, Effect of Glc and Suc on Arabidopsis survival after anoxia. Arabidopsis seeds were germinated under aerobic conditions for 4 d on vertical agar plates containing Murashige and Skoog medium (0.5×) in presence or absence of exogenous Suc or Glc (90 mm). Plates were subsequently transferred to anoxia for 3 d, and recovery of seedlings was observed after 2 additional weeks of aerobic growth in the light.

Figure 3.

Validation of the anoxia-inducibility of genes selected on the basis of microarray analysis. A, Arabidopsis seeds were germinated under aerobic conditions for 4 d. Seedlings were kept under aerobic conditions (Air) or transferred to anoxia (Anoxia) for 1 to 72 h. B, Arabidopsis seeds were germinated under aerobic conditions for 4 d and subsequently transferred to anoxia for 10 to 60 min. RNA was extracted, electrophoresed, and northern analysis carried out using gene specific probes. Equal loading was checked by reprobing with a rRNA probe (not shown). A representative experiment is shown.

Figure 4.

Effects of anoxia on carbohydrate metabolism, glycolysis, and mitochondrial respiration. A, Arabidopsis seeds were germinated under aerobic conditions for 4 d and subsequently transferred to anoxia for 6 h. Microarray data (averaged changes in transcript level from two biological replicates) were analyzed using the MapMan software. The output of the software is shown, with the genes involved in each metabolic step represented by a small square. A blue square (and blue shades) indicates a gene whose transcript level increased following the anoxic treatment. A red square (and red shades) indicates a gene whose transcript level decreased following the anoxic treatment. A color scale was used, in which a pale coloration represents a 1-fold change in expression, and the response saturates at a 4-fold change. Arabidopsis Genome Initiative code colored in blue/red indicates a stronger/reduced anoxia response, respectively, in the presence of exogenous Suc. B, Effects of Suc on the expression of two anoxia-inducible Suc synthase genes. Suc synthase genes significantly affected by exogenous Suc were identified by comparing the datasets (air versus air + Suc; air versus anoxia; air versus anoxia + Suc) by using the Microarray Analysis Suite 5.0 software algorithm. The fold-change measured in two independent experiments (EXP. 1 and EXP. 2) is shown. C, Time course of the expression of SUS1 in presence/absence of Suc. RNA was extracted, electrophoresed, and northern analysis carried out using a gene-specific probe. Equal loading was checked by reprobing with an rRNA probe (not shown). A representative experiment is shown. D, Effects of Suc on the expression of a Suc-P-synthase gene (At5g11110). This Suc-P-synthase gene was identified by comparing the datasets (air versus air + Suc; air versus anoxia; air versus anoxia + Suc) by using the Microarray Analysis Suite 5.0 software algorithm. The fold-change measured in two independent experiments (EXP.1 and EXP. 2) is shown.

Figure 5.

Effects of Suc on the anoxic transcriptome. A, Scatter plot showing the correlation between gene modulation under anoxia and under anoxia with exogenous Suc (ANOXIA + SUC). Expression data were filtered to select only genes showing a coincident change-call in both the replicates and for each experimental condition. Furthermore, only replicates whose ±sd of the averaged signal log₂ ratio did not exceed 50% of the mean value were selected. Genes significantly affected by exogenous Suc were identified by comparing the datasets (anoxia versus anoxia + Suc) by using the Microarray Analysis Suite 5.0 software algorithm. Only genes showing a significant change call in both the replicates were selected and are shown as blue and red dots in the scatter plot. Blue dots identify genes whose anoxia-modulation was enhanced by Suc, while red dots identify genes showing a reduction of the anoxic response as a consequence of Suc addition to the incubation medium. B, Genes significantly repressed by anoxia and annotated as coding for proteins involved in auxin physiology were selected, and their change of expression (fold change) under anoxia was plotted against the anoxia + Suc fold change. C, Genes significantly induced by anoxia and annotated as coding for HSP were selected, and their change of expression (fold change) under anoxia was plotted against the anoxia + Suc fold change. As a reference for the overall effects of Suc on gene induction, the regression line of data reported in Figure 5A is shown as black dotted line. The data reported are mean of two biological replicates ±sd did not exceed 50% of the mean value. D, Effect of Suc, anoxia, and anoxia + Suc on the transcript level of genes coding for HSP. Only genes showing a clear response to presence of Suc under anoxia are listed. Data are expressed as average fold change from two biological replicates for each experimental condition. E, Effect of heat pretreatment on anoxia survival of Arabidopsis seedlings. One-week-old seedlings were used, to allow an easy visualization of the root system. Seedlings were treated at 38°C for 1.5 h immediately before the 6-h anoxia treatment. Exogenous Suc was added to the growing medium at the concentration shown in figure. A representative experiment is shown.