Jasmonate

Abstract

Under stress conditions that bring about excessive absorption of light energy in the chloroplasts, the formation of singlet oxygen (¹O₂) can be strongly enhanced, triggering programmed cell death. However, the ¹O₂ signaling pathway can also lead to acclimation to photooxidative stress, when ¹O₂ is produced in relatively low amounts. This acclimatory response is associated with a strong downregulation of the jasmonate biosynthesis pathway and the maintenance of low jasmonate levels, even under high light stress conditions that normally induce jasmonate synthesis. These findings suggest a central role for this phytohormone in the orientation of the ¹O₂ signaling pathway toward cell death or acclimation. This conclusion is confirmed here in an Arabidopsis double mutant obtained by crossing the ¹O₂-overproducing mutant ch1 and the jasmonate-deficient mutant dde2. This double mutant was found to be constitutively resistant to ¹O₂ stress and to display a strongly stimulated growth rate compared with the single ch1 mutant. However, the involvement of other phytohormones, such as ethylene, cannot be excluded.

The sedentary nature of terrestrial plants obliges them to compensate their reduced mobility by a high plasticity in order to rapidly and efficiently adjust their metabolism and maintain their growth performance in a wide range of environmental conditions. Acclimation of plants to environmental challenges involves a variety of genetically controlled physiological and metabolic changes that allow the organism to cope with the new environment. Among the main environmental factors that can affect agricultural crop productivity, sunlight appears to be one of the most crucial and intensively studied, revealing a large variety of responses occurring in different time ranges from rapid biophysical adjustments to slower changes in gene expression and reorganization of the photosynthetic apparatus.^1-4 A major problem occurs when light energy is absorbed by plants in excess of what can be used by the photosynthetic processes, e.g., when the photosynthetic activity is inhibited by stress. Excess excitation/electron can leak to O₂, leading to the formation of reactive O₂ species (ROS).⁵ In particular, under high light stress, conversion of singlet excited chlorophylls (¹Chl*) to the triplet excited state (³Chl*) by intersystem crossing is favored and, because of its relatively long lifetime, ³Chl* can react with O₂ to form singlet oxygen (¹O₂) by triplet annihilation.⁶ This ROS is toxic, causing damage to macromolecules,^7,8 and is believed to play a major destructive role during the execution of ROS-induced cell death in leaf tissues.^9,10

A recent study performed on the chlorina1 mutant (ch1) of Arabidopsis thaliana has shown that vascular plants have the capacity to acclimate to ¹O₂.¹¹ A similar phenomenon was previously reported in the microalga Chlamydomonas.¹² The Arabidopsis ch1 mutant is characterized by a lack of chlorophyll b, leading to a complete deficiency in PSII chlorophyll-protein antenna complexes (LHCII) and to PSII units restricted to their reaction centers.¹³ This mutant is highly photosensitive,^9,13 due to a selective increase in the release of ¹O₂ by the “naked” PSII centers, as measured by different techniques including ¹O₂-specific fluorescent probes, EPR spectroscopy of spin traps, HPLC analyses of ¹O₂ lipidic markers, and gene expression profiles.^11,14 As illustrated in Figure 1, increasing photon flux density (PFD) from 200 μmol photons m^–2 s^–1 (growth conditions) to 1000 μmol m^–2 s^–1 caused leaf bleaching (Fig. 1A), lipid peroxidation (Fig. 1B and C) and loss of PSII activity (Fig. 1D) in ch1 plants. These effects were not observed in the wild type under similar conditions (not shown).

Figure 1. Photooxidative stress in the ch1 single mutant and the ch1 dde2 double mutant exposed for 27 h to high light (1000 µmol photons m^–2 s^–1) and low temperature (10°C). (A) Plants after the light/cold treatment. (B) Autoluminescence imaging of lipid peroxidation after the light/cold treatment. Color scale indicates signal intensity from 0 (black) to saturation (white); (C) ROS-induced lipid peroxidation (HOTE levels) before (white bars) and after the treatment (black bars). Data are mean values of 3 separate experiments + SD (D) PSII photochemical efficiency (Fv/Fm) after the treatment. The plot represents the % of leaves in 3 classes of Fv/Fm values.

Pre-exposure to a moderately elevated PFD (e.g., 400 μmol m^–2 s^–1) for a few days, which induced a low production of ¹O_2, was shown to markedly enhance the tolerance of ch1 plants to photooxidative stress.¹¹ This increased phototolerance was not associated with a decreased rate of ¹O₂ formation and was accompanied by the induction of many genes, particularly in the categories related to metabolism, oxidative stress responses and regulation of transcription.¹¹ Thus, moderately elevated PFDs oriented the ¹O₂ signaling pathway toward acclimation and prevented ¹O₂-induced programmed cell death (PCD). A striking phenomenon associated with this acclimation process was a downregulation of the jasmonate (JA) biosynthesis pathway, with most of the genes involved in this pathway being strongly repressed.¹¹ This was accompanied by a very low concentration of JA in the acclimated leaves, which contrasted with the marked stimulation of JA synthesis in non-acclimated leaves exposed to high light stress. A block of JA synthesis during acclimation to ¹O₂ is consistent with previous data obtained on the Arabidopsis flu mutant¹⁵ that have shown that JA accumulates and promotes cell death under ¹O₂ stress.^16-18 This is also consistent with previous observations showing that leaf tissue or suspension-cultured cells undergo PCD upon exogenous addition of methyl JA.¹⁹ Actually, the involvement of JA in the regulation of PCD in plants has been reported in various plant species in response to different stressors.^20,21 In the flu mutant, the JA precursor, 12-oxo phytodienoic acid (OPDA), was found to antagonize the JA activity.¹⁸ However, OPDA did not increase in ch1 leaves under light stress or under acclimatory conditions,¹¹ suggesting that modulation of the sensitivity to ¹O₂ in ch1 was not attributable to changes in OPDA levels.

To further study the role of JA biosynthesis in the acclimation of the ch1 mutant to ¹O₂, we crossed this mutant with the dde2 Arabidopsis mutant (delayed-dehiscence 2) to create a ¹O₂-producing mutant deficient in JA. The dde2 mutant is unable to metabolize 13-hydroperoxy linolenic acid (HOTE) and to synthesize JA, due to the breakdown of the gene encoding allene oxide synthase.²² The ch1 dde2 double mutant was noticeably more resistant to high-light stress than the ch1 single mutant (Fig. 1). Indeed, we observed a marked attenuation of leaf bleaching after a 27 h-treatment under a high light regime (Fig. 1A) and a lower level of lipid peroxidation in the ch1 dde2 mutant compared with the single mutant ch1, as imaged by plant autoluminescence (Fig. 1B) or indicated by the HOTE levels (Fig. 1C). Moreover, after high light stress, the chlorophyll fluorescence parameter Fv/Fm was < 0.4 in most ch1 leaves while the majority of ch1 dde2 leaves had a Fv/Fm ratio above 0.4 (Fig. 1D). This indicates less inhibition of PSII photochemical efficiency in the double mutant. Clearly, inhibition of JA synthesis is beneficial to plants exposed to ¹O₂ stress, precluding the development of PCD. Accordingly, applications of exogenous JA to acclimated ch1 plants cancelled the increased tolerance to ¹O₂ stress.¹¹ The initial production of ¹O₂ linked to the ch1 mutation should be equivalent in both mutants, and therefore the maintenance of low levels of lipid peroxidation in the absence of JA can be explained by a block of PCD and the activation of defense and detoxification mechanisms, limiting oxidative damage.

Strikingly, when grown under standard conditions of light, the double mutant ch1 dde2 was also characterized by a much higher growth rate compared with the single mutant ch1 (Fig. 2), suggesting a negative effect of JA on growth, even in the absence of high light stress. The effect was spectacular with almost a doubling of the size of the JA-deficient ch1 plants compared with the JA-producing plants. Analyses of ¹O₂-specific oxidation products of linolenic acid and β-carotene have shown that ¹O₂ is produced in plant leaves even in low light, resulting in a chronic oxidation of lipids and carotenoids, and this phenomenon is exacerbated in the ch1 mutant due to the enhanced formation of ¹O₂.^9,23 JA is an essential plant hormone that regulates defense responses and development processes.^21,24 Moreover, it is known that JA and light signaling are interconnected for the regulation of growth and development, with their interaction determining the metabolic balance between growth and defense.²⁵ Our observation that JA synthesis regulates negatively plant growth under conditions of chronic ¹O₂ formation supports those previous conclusions.

Figure 2. Growth characteristics of the ch1 single mutant and the ch1 dde2 double mutant. (A) Plants aged 49 d. (B) Growth curve (rosette diameter) of plants. Plants were grown under standard conditions: 200 µmol photons m^–2 s^–1; 8 h photoperiod; 20/18°C, day/night temperature. Data are mean values of 7 and 14 measurements (ch1 and ch1 dde2, respectively) +SD.

The findings described above suggest a central role for jasmonate in the ¹O₂ signaling networks and in the orientation of plant metabolism toward PCD or acclimation to ¹O₂. JA appears to be one of the decision makers in this choice, with the increased synthesis of this hormone favoring the “growth arrest/cell death” options (Fig. 3). The jasmonate pathway has been predicted in Chlamydomonas,^26,27 and therefore it would be interesting to check the link between jasmonate and ¹O₂ acclimation in this species. However, we cannot exclude the involvement of other phytohormones in this process. Although the concentrations of both OPDA and salicylic acid remain stable in ch1 leaves under various light conditions,¹¹ microarray-based transcriptomic analyses revealed the induction of genes related to ethylene such ACS2 (1-aminocyclopropane-1-carboxylic acid synthase) and ERF (ethylene response factor) during ¹O₂ stress. No such induction was observed during acclimation.¹¹ Induction of ethylene synthesis by high light is documented in photosynthetic organisms.²⁸ Therefore, one cannot exclude the involvement of both JA and ethylene in the regulation of ¹O₂-induced cell death. This interaction was actually reported in tomato for cell death induced by a toxin.²⁹

Figure 3. Schematic representation of ¹O₂ signaling in the ch1 mutant under (A) acclimatory or (B) photooxidative stress conditions.

Oxylipin hormones are known to function as regulators of defense against herbivores and pathogens.^30,31 Therefore, as recently pointed out by Demmig-Adams et al.,³² the inhibition of jasmonate synthesis in plants acclimated to photooxidative stress may have negative effects on the tolerance to biotic stresses. Accordingly, Arabidopsis mutants deficient in lipid-soluble antioxidants were found to accumulate oxylipin hormones and to have superior defenses against insect herbivores.³³ Thus, there could be a compatibility issue between tolerance toward abiotic stresses and defense against biotic stresses. This important point deserves to be addressed in future research.

Glossary

Abbreviations:

¹O₂

singlet oxygen

Chl

chlorophyll

PSII

photosystem II

ROS

reactive oxygen species

PFD

photon flux density

PCD

programmed cell death

JA

jasmonic acid

10.4161/psb.26655

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.