Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis

Abstract

In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H(2)O(2)) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H(2)O(2). Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H(2)O(2) stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H(2)O(2) was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H(2)O(2) transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H(2)O(2)-deregulated genes, positioning elevated H(2)O(2) levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes encoding unknown proteins.

Figure 1.

Hierarchical average linkage clustering of 1,495 differentially expressed genes in control and catalase-deficient Arabidopsis plants during HL irradiation. Temporal expression patterns in control and catalase-deficient (CAT2HP1) plants during the HL time course (0, 3, and 8 h) are presented. Yellow and blue correspond to up-regulation and down-regulation, respectively. Four main clusters of transcriptional changes (A, B, C, and D) are indicated.

Figure 2.

Verification of microarray results by cDNA-AFLP. Expression profiles obtained by cDNA-AFLP are shown for 13 transcripts whose similar expression patterns were found in the microarray analysis. For the sake of clarity, three different graphs (A, B, and C) group transcripts with similar kinetics during the HL treatment. For control and CAT2HP1 plants, each color line connects the different time points at which the samples were collected. Arabidopsis Genome Initiative (AGI) codes and annotations are indicated. Expression data were processed as described in “Materials and Methods.”

Figure 3.

Differentially regulated genes in CAT2HP1 under ambient growth conditions: temporal expression patterns during HL and their responsiveness to various environmental stimuli. The 30 most up-regulated (A) or down-regulated (B) genes in nonchallenged CAT2HP1 plants are presented. Hierarchical average linkage clustering of the genes during 0, 3, and 8 h of HL irradiation in CAT2HP1 and control plants (left) and corresponding response profiles to various stress conditions and treatment with plant hormones (right) are shown. AGI codes and annotations are indicated. Data were obtained with genevestigator, a database and Web browser data mining interface for Affymetrix GeneChip data, allowing a rapid assessment of the expression characteristics of selected genes throughout a wide variety of environmental and developmental conditions (Zimmermann et al., 2004). ACC, 1-Aminocyclopropane-1-carboxylic acid; GA3, gibberellic acid 3; ABA, abscisic acid; BL, brassinolide; IAA, indole-3-acetic acid.

Figure 4.

H₂O₂-up-regulated genes within three principal environmental stresses. The different stress conditions (cold, heat, and drought) are indicated together with the number of genes within the overlap with the H₂O₂-responsive genes and the total number of gene input of each of the stresses as well. In the Venn diagram, numbers of genes are given that are unique to that gene set or in the common sections between sets, with the amount of transcription factors present within a specific gene set in parentheses. AGI codes and descriptions are given for genes in the common sections of the stresses. Transcription factors are indicated in bold.

Figure 5.

Induction of HSPs in catalase-deficient plants during HL stress. Hierarchical average linkage clustering of the HSPs and HSFs induced during 0, 3, and 8 h of HL irradiation in the control and CAT2HP1 plants. Only HSPs/HSFs with a more than 3-fold increase in expression are included. AGI codes and annotations are indicated.

Figure 6.

Schematic overview of the flavonoid biosynthetic pathway in Arabidopsis (based on Winkel-Shirley, 2002; Kitamura et al., 2004). Catalyzing enzymes, AGI codes, and corresponding genetic loci in parentheses are indicated. Graphs represent the expression profiles of the corresponding genes in response to 0, 3, and 8 h of HL irradiation: solid line, control; dashed, CAT2HP1. Dashed arrows indicate different branches of the flavonoid pathway involved in the biosynthesis of other flavonoids or secondary metabolites that were not activated in the experiment. The names of the major classes of (flavonoid) end products are boxed. C4H, Cinnamate-4-hydroxylase; ACC, acetyl-CoA carboxylase; CHI, chalcone isomerase; F3H, flavonone 3-hydroxylase; F3′H, flavonoid 3′ hydroxylase; DFR, dihydroflavonol reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; UFGT, UDP-Glc:flavonoid glycosyltransferase; GST, glutathione S-transferase; TT, testa transparent; DIN, dark-inducible.

Figure 7.

Quantification of anthocyanin content. Bars represent the mean anthocyanin content (expressed as absorbance per g FW) in control and CAT2HP1 plants exposed to 0 and 24 h of HL irradiation. Error bars correspond to ±se.

Figure 8.

Hierarchical average linkage clustering of the identified anthocyanin cluster and its behavior during senescence. Temporal expression patterns in control and CAT2HP1 plants during the HL time course (0, 3, and 8 h) are presented together with their expression profile during senescence. Two main clusters of transcriptional changes (A and B) are indicated.