A genetic framework for H2O2 induced cell death in Arabidopsis thaliana

Abstract

Background: To survive in a changing environment plants constantly monitor their surroundings. In response to several stresses and during photorespiration plants use reactive oxygen species as signaling molecules. The Arabidopsis thaliana catalase2 (cat2) mutant lacks a peroxisomal catalase and under photorespiratory conditions accumulates H2O2, which leads to activation of cell death.

Methods: A cat2 double mutant collection was generated through crossing and scored for cell death in different assays. Selected double mutants were further analyzed for photosynthetic performance and H2O2 accumulation.

Results: We used a targeted mutant analysis with more than 50 cat2 double mutants to investigate the role of stress hormones and other defense regulators in H2O2-mediated cell death. Several transcription factors (AS1, MYB30, MYC2, WRKY70), cell death regulators (RCD1, DND1) and hormone regulators (AXR1, ERA1, SID2, EDS1, SGT1b) were essential for execution of cell death in cat2. Genetic loci required for cell death in cat2 was compared with regulators of cell death in spontaneous lesion mimic mutants and led to the identification of a core set of plant cell death regulators. Analysis of gene expression data from cat2 and plants undergoing cell death revealed similar gene expression profiles, further supporting the existence of a common program for regulation of plant cell death.

Conclusions: Our results provide a genetic framework for further study on the role of H2O2 in regulation of cell death. The hormones salicylic acid, jasmonic acid and auxin, as well as their interaction, are crucial determinants of cell death regulation.

Fig. 1

Survival of cat2 and cat2 double mutants in the in vitro assay. Fourteen-day old seedlings were transferred from SD (8 h/16 h) to LD (12 h/12 h) and gas exchange was restricted by sealing of plates with parafilm. Survival was scored after 8 days. Asterisks indicate significant difference from cat2 single mutant at the end of LD treatment (n = 30; p <0.05, Fisher LSD). For a version of this figure with a time course of survival, see Additional file 2: Figure S1

Fig. 2

Formation of lesions in soil-grown cat2 and cat2 double mutants. Selected double mutants without lesions (cat2 axr1, cat2 era1, cat2 rcd1, cat2 as1), with few lesions (cat2 npr1, cat2 sid2, cat2 sgt1b, cat2 rar1) and with more lesions than cat2 (cat2 jar1, cat2 bak1, cat2 wrky25). Cell death is indicated by trypan blue stain of 4 weeks old plants. White scale bar shows 1 cm and black scale bar 600 μm. All mutants used in this study are in Additional files 3, 4 and 5: Figures S2–S4

Fig. 3

Maximum photosynthetic efficiency (F_v/F_m) in dark adapted plants after transfer to continuous light and restricted gas-exchange. Fourteen-days old seedlings were transferred from 70 μmol m⁻² s⁻¹ light intensity and 8 h day/16 h night to 120 μmol m⁻² s⁻¹ continuous light. F_v/F_m was measured on shift day, 2^nd, 4^th, and 7^th. The experiment was repeated three times using 20 plants per repeat (n = 60). Asterisks indicate significantly different F_v/F_m values of double mutant in time scale and compared to cat2 (p <0.05, General Linear Model)

Fig. 4

H₂O₂ concentration in cat2 and selected double mutants in 4 weeks old plants measured with Amplex Red. Letter “a” indicates significant difference from Col-0 and letter “b” significant difference from cat2 (mean ± SE, n = 6)

Fig. 5

Lesion development in soil-grown cat2 double and triple mutants. Cell death is indicated by trypan blue stain for weeks old plants. White scale bar shows 1 cm and black scale bar 600 μm

Fig. 6

Cluster analysis of cell death related genes in cat2 and other LMMs. Experiments performed on the Affymetrix ATH1 chip were obtained from cat2, LMMs, constitutive defense mutants, hormone treatments, senescence and biotic and abiotic stresses (see Methods for a full list of experiments). The gene ontology category cell death (488 genes) was used for Bayesian hierarchal clustering. Values are mean of log2 ratio of the treatment and control expressions. Magenta and green indicate increased and decreased expression compared with untreated or wild type plants, respectively

Fig. 7

Local gene expression in cat2. Histochemical analyses of uidA expression in Col-0 and cat2 in 4 week old soil-grown plants. Promoter-uidA lines included PR1, PDF1.1, DR5 and FMO1 (Table 2)

Fig. 8

Model for ROS in cell death regulation. Intracellular H₂O₂ production caused by the cat2 mutation lead to increased concentration of hormones (SA and JA, through SID2 and JAR1) and activation of multiple signaling pathways. The balance and timing of biosynthesis of auxin, SA and JA is one important determinant of cell death. In parallel, MPK signaling and other unknown signaling pathways may directly target TFs and regulate their activity and interaction with DNA with a subsequent change in gene expression. Other mechanisms in the nucleus also provide a suitable environment for accurate gene expression, including RCD1 that interacts with TFs. A yet to be identified regulator of cell death is farnesylated for proper function by ERA1. Extensive cross-talk between hormone signaling pathways allow fine tuning of the signal. Controlled ROS production via BAK1 and RBOHs may provide a signal to neighboring cells leading to propagation of cell death