Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses

Abstract

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.

Figure 1.

Growth suppression of double mutant apx1/tylapx. A, Protein blots showing the absence of cytosolic APX1 and the decreased expression of tylAPX in the different mutants and the double mutant grown under controlled growth conditions. B, Fresh weights of 17-d-old plants grown under controlled conditions, presented as an average of 10 individual plants per line. **, P = 0.01 in t test. The single mutants APX1/tylapx, apx1/tylAPX, and the double mutant apx1/tylapx are presented next to their respective wild-type controls, and the genetic background of each line is given in parentheses.

Figure 2.

Differences in time of bolting between the different APX mutants and the double mutant. A to C, Inflorescence length was measured for each mutant during growth under controlled conditions. The different mutant lines are presented with respect to their corresponding controls. Genetic backgrounds are shown in parentheses. D, Number of leaves for all lines measured during growth under controlled conditions is given as a control. ses are given for 40 replicates of each line. Controlled growth conditions: 22°C to 23°C, continuous light, 40 μmol m⁻² s⁻¹, and a relative humidity of 70%.

Figure 3.

Suppressed level of protein oxidation in the double mutant (apx1/tylapx) in response to light stress. Plants were grown under continuous low light conditions (25 μmol m⁻² s⁻¹) and transferred to moderate light (300 μmol m⁻² s⁻¹). Detection of proteins containing carbonyl groups (indicative of protein oxidation) was performed by a protein-blot assay (top). The expression level of APX proteins in the different mutants was determined by a protein blot (second segment from the top). Rubisco protein level was also determined by protein blots (rbcL and rbcS) and is used to demonstrate equal loading (bottom). Measurements were performed as described in “Materials and Methods.”

Figure 4.

Suppressed expression of the zinc-finger protein Zat12 in apx1/tylapx in response to light stress. Plants were grown under continuous light (40 μmol m⁻² s⁻¹) and exposed to high light intensity (900 μmol m⁻² s⁻¹). At different times, plant tissue was collected from the different mutants and processed for RNA blots. Blots were probed with different cDNAs encoding proteins involved in ROS production (RbohD) or scavenging (APXs and peroxiredoxin Q [Prx-Q]).

Figure 5.

Enhanced accumulation of anthocyanin in apx1/tylapx in response to light stress. Anthocyanin levels were measured in shoots of 21-d-old wild-type and mutant plants exposed to light stress (900 μmol m⁻² s⁻¹) for 24 and 48 h. *, P = 0.05; **, P = 0.01 in t test.

Figure 6.

Differences in abiotic stress tolerance among apx1, tylapx, and apx1/tylapx mutants. Effects of abiotic stresses imposed by paraquat (A), salinity (B), osmotic stress (sorbitol; C), and heat stress (38°C; D) on root elongation in 5-d-old seedlings of the different lines and their corresponding controls were measured with seedlings grown on agar plates. All lines germinated simultaneously with a germination rate of 97% to 100%. Measurements were performed as described in “Materials and Methods.” *, P = 0.05; **, P = 0.01 in t test.

Figure 7.

Enhanced basal thermotolerance of tylapx and apx1/tylapx mutants. Basal thermotolerance was measured by scoring the survival rate of 5-d-old seedlings of the different lines and their corresponding controls subjected to a 2-h heat treatment (45°C). No differences were found among the different lines and their corresponding controls in acquired thermotolerance. All lines germinated simultaneously with a germination rate of 97% to 100%. **, P = 0.01 in t test.

Figure 8.

Impaired basal thermotolerance of mutants deficient in retrograde signaling. Survival rates were scored for 5-d-old seedlings of different mutants impaired in chloroplast-to-nuclei retrograde signaling (gun1, gun5, and abi4), subjected to a 2-h heat treatment (45°C; basal thermotolerance), or treated at 38°C for 1.5 h, allowed to recover for 1 h at 21°C, and subjected to 45°C for 2 h (acquired thermotolerance). All lines germinated simultaneously with a germination rate of 97% to 100%. **, P = 0.01 in t test.

Figure 9.

Venn diagram summarizing the different pathways affected in apx1, tylapx, and apx1/tylapx mutants. The diagram illustrates the complex interactions between the pathways affected in plants by the absence of APX1, tylAPX, and/or APX1 and tylAPX and includes developmental effects on growth and flowering, differences in expression of defense pathways, and tolerance to different abiotic stresses.