Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes

Abstract

The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxide-responsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAAAAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H(2)O(2). Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves.

Fig. 1

Superoxide generation by MV treatment in illuminated Arabidopsis seedlings. Two-week-old Arabidopsis seedlings grown on soil were subjected to 50 μM MV treatment in the light (120 μmol photons m⁻² s⁻¹) for the duration indicated below each panel. Leaf samples were stained for O₂^•− accumulation by the NBT staining after MV treatment, and a complete Arabidopsis seedling is also shown. Generation of H₂O₂ after identical MV treatment were followed by DAB staining and the results are shown in the right panels

Fig. 2

GS activity and Western blot analysis of GS, Rubisco, Hsp70 and 2-Cys Prx after oxidative treatment of Arabidopsis leaves. Time courses of (a) GS activity (25 μg), (b) GS protein (80 μg), (c) Rubisco large subunit (LSU) (10 μg), (d) cytoplasmic (cyt) and (e) chloroplastic (chl) Hsp70 (80 μg), (f) 2-Cys Prx (15 μg) in total leaf extracts following 50 μM MV plant treatment with light. Leaf protein extracts (7 μg) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (g). Arrows indicate: cytosolic (GS1) and chloroplastic (GS2) GS isoforms (panel b), Rubisco large subunit degradation product (panel c) and 2-Cys Prx dimer (d) and monomer (m) (panel f)

Fig. 3

Analysis of MV up-regulated genes by Northern blot hybridization. Twenty micrograms of total RNA from control and MV-treated seedlings harvested at the indicated times after the onset of MV treatment were fractionated on formaldehyde–agarose gels and transferred to nylon membranes. Membranes were hybridized with ³²P-labelled cDNA fragments of the indicated genes and their MIPS identifiers are indicated

Fig. 4

Sequence logos of relevant motifs conserved in promoters of MV induced genes. The relative height of each letter indicates the relative abundance of the corresponding nucleotide at that position in each motif

Fig. 5

DNA binding activity of protein extracts of MV-treated Arabidopsis leaves. (a) Competition experiments were carried out with Arabidopsis leaf protein extracts and a ³²P-labelled W box probe. As competitors, 100-fold unlabelled W box or mW box probes were added. Arrows indicate specific retarded bands. (b) DNA binding activity of Arabidopsis leaf protein extracts from control or MV-treated leaves. (c) Nucleotide sequences of probes used for the DNA binding assays. W box contains a double W box (AGTCAA), which was mutated into AGGTCA in the mW box. FP: free probe

Fig. 6

Response of WRKY30 expression to oxidative stress in Arabidopsis leaves. PrAtWRKY30::GUS lines were exposed to 50 μM MV or 20 mM H₂O₂ for the times indicated. A control leaf is shown in the right panel

Fig. 7

ROS-regulated genes within oxidative and high light conditions in Arabidopsis. Wild-type plants treated with 20 mM H₂O₂ for 1 h (Davletova et al. 2005), 50 μM MV for 2 h (present work) and CAT- deficient Arabidopsis mutant subjected for 3 h to high light (HL) (Vanderauwera et al. 2005) are indicated together with the total number of gene input of each stress condition. For comparison, genes whose expression ratios varied at least 3-fold between treated and control samples were considered. In the Venn diagram, numbers of genes are given that are unique to that gene set or in common sections between sets

Fig. 8

Hierarchical clustering of combined abiotic stress data sets with the identified 2 h MV up-regulated genes. Clusters of early and late responsive genes as well as a group of unresponsive genes are indicated. Locus identification numbers are shown for representative clusters containing functionally related genes involved in cold (C), drought (D), genotoxic (G), heat (H), osmotic (Os), oxidative (Ox), salt (S), and wounding (W). I, II and III indicate clusters of early transcriptional changes and IV a group of genes responsive to 2 h MV but not to any other environmental stress. CL means cold late. Red and green indicate up- and down-regulation, respectively. The full-color range represents log base 2 ratios of −2.0 to 2.0