Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis

Abstract

Plants have evolved adaptation mechanisms to sense oxygen deficiency in their environments and make coordinated physiological and structural adjustments to enhance their hypoxic tolerance. To gain insight into how plants respond to low-oxygen stress, gene expression profiling using whole-genome DNA amplicon microarrays was carried out at seven time points over 24 h, in wild-type and transgenic P(SAG12):ipt Arabidopsis (Arabidopsis thaliana) plants under normoxic and hypoxic conditions. Transcript levels of genes involved in glycolysis and fermentation pathways, ethylene synthesis and perception, calcium signaling, nitrogen utilization, trehalose metabolism, and alkaloid synthesis were significantly altered in response to oxygen limitation. Analysis based on gene ontology assignments suggested a significant down-regulation of genes whose functions are associated with cell walls, nucleosome structures, water channels, and ion transporters and a significant up-regulation of genes involved in transcriptional regulation, protein kinase activity, and auxin responses under conditions of oxygen shortage. Promoter analysis on a cluster of up-regulated genes revealed a significant overrepresentation of the AtMYB2-binding motif (GT motif), a sugar response element-like motif, and a G-box-related sequence, and also identified several putative anaerobic response elements. Finally, quantitative real-time polymerase chain reactions using 29 selected genes independently verified the microarray results. This study represents one of the most comprehensive analyses conducted to date investigating hypoxia-responsive transcriptional networks in plants.

Figure 1.

Induced expression of adh1 gene in wild-type plants in response to low oxygen. White bars represent the level of adh1 transcript measured using qRT-PCR in plants exposed to air of ambient composition, whereas black bars designate the level of adh1 transcript in samples treated with 3% oxygen for the duration of time indicated on the graph.

Figure 2.

Differentially expressed genes identified using SAM. The number of PSGs and NSGs for wild-type (A) and transgenic P_SAG12:ipt plants (B) are represented with black bars above and white bars below the x axis, respectively, for the time points indicated. The hashed area within each bar designates the number of significant genes within the category whose expression changed by at least 2-fold in response to the low-oxygen treatment.

Figure 3.

Representation of expression changes for differentially expressed genes. Temporal patterns of expression in wild-type and transgenic P_SAG12:ipt plants during the time course of hypoxic stress (pretreatment, posttreatment, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) were visualized using average HLC (left) and KMC (right), both with a Euclidian distance metric. The color of each element in the left section represents the expression level of each gene as log₂(hypoxia/normoxia), with blue representing down-regulation and yellow representing up-regulation. The color of individual lines in the right section represents temporal profiles in each of the eight clusters corresponding to the gene groups highlighted in the hierarchical clustering dendrogram; the pink lines represent the average expression of each group.

Figure 4.

Hypoxia-induced and suppressed functional classes and pathways. Selective GO terms and KEGG pathways overrepresented in positively (A) or negatively (B) significantly expressed gene classes identified by EASE are presented as heat maps. Here, minus log₁₀ transformed P values [−log₁₀(P)] and log₁₀ transformed P values [log₁₀(P)] were presented in A and B, respectively, for up- and down-regulated genes. BP, Biological process; MF, molecular function; CC, cellular component; KP, KEGG pathway.

Figure 5.

Sequence motifs overrepresented in hypoxia-induced promoters. Each sequence was individually identified as significantly enriched in the selected subset. Sequences representing the same motif are aligned in subgroups. The P value was determined using the binomial distribution (Hudson and Quail, 2003) representing the likelihood of the observed occurrences in a randomly selected set of promoters. Footnotes: a, Hoeren et al. (1998); b, Sun et al. (2003); and c, Guiltinan et al. (1990), Hong et al. (1995).

Figure 6.

Correlation of gene expression values measured by microarrays and qRT-PCR. Changes in gene expression for 29 genes represented as log₂(hypoxia/normoxia) derived from qRT-PCR and microarray hybridizations were compared. Each gene had two pairs of values, which are also listed in Table I.