Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

Abstract

Background: Reactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.

Results: To investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling.

Conclusions: We propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.

Figure 1

Effect of high light treatment on plant growth and on gene expression at low temperature. A) Phenotype of WT and npq1lut2 plants at time 0 and 24 h under the high-light and low temperature conditions specified in the text. B) Number of genes (probe set) up or down regulated in npq1lut2 mutant compared to control wild-type plants at the three time points of the experiment (0, 2 and 24 hours of treatment at 10°C and 1000 μmol photons m^-2 s^-1). White panels are genes from at least 1-fold change to 2-fold change difference, grey panels are genes from at least 2-fold change to 3-fold change difference and black panels are genes with at least a 3-fold change between the mutant and the control. Fold change is indicated in a log₂ scale.

Figure 2

Steady-state accumulation of ROS species and protein oxidizing activity in wild type and npq1lut2 mutant plants. Specific probes were used to quantify the accumulation of several ROS in wild type and npq1lut2 detached leaves under stress (1000 μmol photons m^-2 s^-1, 10°C). (A) DFC and (B) ProxF fluorescence was used to follow the accumulation of reduced forms of ROS. (C) SOSG fluorescence was used to follow singlet oxygen (¹O₂). Details on ROS measurements are given in material and method session. Symbols and error bars show means ± SD. (D) Western-blots were used to detect oxidized thylakoid proteins extracted from wild type and npq1lut2 membranes. WT and npq1lut2 rosettes were pre-treated for 48 h at 10°C and low light as described in methods, then were exposed to photoxidative conditions (1000 μmol photon m^-2 s^-1, 10°C, 16 h light/8 h dark). Leaves were harvested and thylakoids isolated before stress (0) and at same time after 1, 2 and 5 days of HL.

Figure 3

Lhc gene expression. Light harvesting complex (Lhc) gene expression after 24 h stress (1000 μmol photons m^-2 s^-1, 10°C) in wild-type (gray bars) and npq1lut2 (white bars) plants. For each sample, the average of three repetitions was used to calculate the fold change, which is expressed using the log₂ scale. The genes significantly down-regulated after RMA analysis are Lhca1 (251814_at), Lhcb1 (255997_s_at; 267002_s_at), Lhcb2 (263345_s_at; 258239_at), Lhcb3 (248151_at), Lhcb4.2 (258993_at), Lhcb6 (259491_at). The genes significantly up-regulated after RMA analysis are Lhcb4.3 (265722_at), Lhcb7 (259970_at), ELIP1 (245306_at) and ELIP2 (258321_at). Lhca4 (252430_at) was significantly down-regulated only in the wild-type plants whereas Lhca6 (256015_at) was significantly up-regulated only in the mutant plants.

Figure 4

Biochemical characterization of thylakoid membrane composition under high light stress. Chlorophylls (A, C), carotenoids (B) and tocopherol (D) content of WT and lut2npq1 plants were measured on leaf acetone extracts as described in "Material and Methods". (E, F) Stoichiometry between photosynthetic pigment-binding complexes under high light stress. PSII/PSI ratio (E) and biochemical antenna size (LHCII/PS ratio, F) were determined by both non-denaturing Deriphat-PAGE and immunoblot-titration using specific antibodies (see "Material and Methods" for details). Symbols and error bars show respectively means ± SD.

Figure 5

Transcriptional induction, dose-dependent to ¹O₂, of genes encoding chloroplast proteins. Most relevant genes encoding chloroplast proteins, that showed a statistically significant response to high light at low temperature only in npq1lut2, are reported. Up-regulation is defined as described in Methods. Abbreviations: ALB, albino; CIA, chloroplast import apparatus; CHY, carotene hyroxylase; DUF, uroporphyrinogen III synthase; GSA, glutamate-1-semialdehyde 2,1-aminomutase; HCF, high chlorophyll fluorescence; HPD, 4-hydroxyphenylpyruvate dioxygenase; LCY, lycopene cyclase; NYE, non-yellowing; PDS, phytoene desaturase; SIG, RNA polymerase sigma subunit; TIC/TOC, translocon of inner/outer chloroplastic membrane; UPM, urophorphyrin methylase; VTE, tocopherol cyclase; ZE, zeaxanthin epoxidase.