Stabilization of thylakoid membranes in isoprene-emitting plants reduces formation of reactive oxygen species

Abstract

Isoprene is emitted by a significant fraction of the world's vegetation. Isoprene makes leaves more thermotolerant, yet we do not fully understand how. We have recently shown that isoprene stabilizes thylakoid membranes under heat stress. Here we show that heat-stressed, isoprene-emitting transgenic Arabidopsis plants also produce a lower pool of reactive oxygen and reactive nitrogen species, and that this was especially due to a lower accumulation of H₂O₂ in isoprene emitting plants. It remains difficult to disentangle whether in heat stressed plants isoprene also directly reacts with and quenches reactive oxygen species (ROS), or reduces ROS formation by stabilizing thylakoids. We present considerations that make the latter a more likely mechanism, under our experimental circumstances.     

Biogenic isoprene emission results sometimes in a very large loss of carbon and energy from plants, especially under stress conditions when carbon uptake is largely inhibited.¹ Thus, it is likely that isoprene biosynthesis provides some benefit to plants to balance the negative selection pressure that would come from such a large loss of energy and carbon. In the search for isoprene function, two theories have received increasing attention. On one side, isoprene was thought to stabilize membranes. This seems to be predominantly related to the lipophylic properties of isoprene and its ability to intercalate into membranes and strengthen them under conditions that make them fragile, especially heat stress.² Earlier research indeed demonstrated that isoprene-emitting plants are more tolerant to heat stress.^3-7 On the other side, isoprene was thought to quench reactive oxygen species (ROS) inside leaves, therefore indirectly providing a general antioxidant action that could be seen under multiple stresses.⁸ This would explain why isoprene also protects leaves from strongly oxidative species such as ozone^9-11 or singlet oxygen.^12,13

Velikova et al.,¹⁴ using several different biophysical techniques, directly assessed the impact of isoprene on thylakoid intactness and functionality. Circular dichroism measurements indicated that isoprene emission allowed macrodomains of thylakoid membranes with embedded photosystem II complexes to remain stable when exposed to temperatures 5–8°C higher in isoprene-emitting Arabidopsis plants than in non-emitting plants. The position of the main thermoluminescence peak (Q_B peak) in isoprene-emitting plants was shifted up by about 10°C suggesting modifications in the lipid environment due to the presence of isoprene. Here ancillary data from this recent work¹⁴ are presented. We show that isoprene-emitting transgenic Arabidopsis plants accumulated less ROS and similar amount reactive nitrogen species (RNS) in comparison to non-emitting wild-type plants, in response to heat stress.

Transgenic plants are great tools to study the impact of single traits on whole plant physiology. Arabidopsis plants were transformed by one of us to introduce the gene encoding for isoprene synthase, the enzyme that make isoprene from its substrate, dimethylallyl diphosphate, DMADP.¹⁵ The amounts of NO and H₂O₂, two key molecules in plant signaling inducing hypersensitive responses to stress,^16,17 were measured in wild-type and IspS plants before stress at growth temperature (22°C) and after exposure to heat (38°C; Fig. 1) for 48 h. A significant amount of research has been recently devoted to unravel the possible interactions of NO with ROS, and how these interactions can modulate the signaling of stress responses.^19,20 Maintenance of physiological ROS levels is essential to ensure an accurate execution of their signaling functions, and to prevent their toxicity. Therefore, plants have evolved an antioxidant system composed of enzymatic and non-enzymatic antioxidants to harmlessly scavenge excessive ROS production.²¹ However, when plants are exposed to unfavorable environmental conditions a rapid increase in ROS and RNS production (the so called “oxidative burst”) is often induced.²² Indeed, a significant increase of H₂O₂, and NO was found in both wild-type and isoprene-emitting heat-stressed Arabidopsis plants (Fig. 1). However, the accumulation of H₂O₂ was substantially higher in wild-types than in IspS lines, whereas NO increased similarly in the two plants compared with pre-stress conditions.

Figure 1. Increase of NO (pink) and H₂O₂ (purple) contents in wild-type (non-emitting) and transgenic IspS (isoprene-emitting) Arabidopsis plants after exposure to heat (38°C for 48 h). Values are percent increase with respect to pools of NO and H₂O₂ detected in wild-type and IspS plants grown at 22°C. Details on measurements of NO emission and calculation of NO concentration inside the leaves, and on determination of H₂O₂ content are described in Velikova et al.¹⁸

By changing the balance between NO and H₂O₂ under heat stress conditions, isoprene emission may also modify the signaling pattern of these molecules.¹⁶ When isoprene is not produced, the simultaneous accumulation of H₂O₂ and NO may trigger cell death signaling, whereas in isoprene-emitting plants the accumulation of H₂O₂ and NO may not reach toxic levels or their ratio may not be adequate. Consequently, the signaling action of the two molecules might be dimmed and hypersensitive responses might be prevented.

Why is only H₂O₂ accumulation reduced in Isps plants? In past experiments with plants naturally emitting isoprene the amount of both H₂O₂⁹ and NO²³ was reduced and it was suggested that isoprene could directly react with ROS and RNS.⁸ However, we note that: (1) NO accumulation was found to be reduced in isoprene-emitting plants compared with isoprene-emitting plants exposed to ozone,²³ but may not be affected in response to a stress that does not involve direct oxidation of the medium, as in our heat stress experiment; (2) NO is not a very reactive molecule and the rate constant of its reaction with isoprene would require a large concentration of isoprene in the leaves for the reaction to occurs.¹ Such a large isoprene concentration may be found in natural, strong emitters but not in transgenic Arabidopsis that emit ten times less than strong emitters¹⁴; (3) the attenuated increase of H₂O₂ in IspS lines may not directly involve H₂O₂ reactivity with, and consequent quenching by isoprene. Lower accumulation of H₂O₂ may be an indirect consequence of preserved functionality of photosynthetic regulatory mechanisms in IspS lines, in turn due to isoprene-induced membrane stabilization.¹⁴ If the photochemical regulatory apparatus is preserved, then the photosynthetic electron transport rate runs more efficiently as we already demonstrated,¹⁴ and photochemical energy is not dissipated through direct oxygen photoreduction.²⁴ Thus the lower H₂O₂ generation might be due to less oxygen photoreduction and consequently less generation of dangerous levels of ROS.²⁵

Isoprene general protection against environmental stresses is well known.⁸ Theoretically, isoprene may both strengthen membranes²⁶ and quench reactive compounds, predominantly reactive oxygen species.⁸ It might be argued that heat stress specifically affects membrane stability, and thus should make isoprene protection particularly evident if isoprene only strengthens membranes. However, the relationship between ROS and membrane stability is shown in a cartoon in Figure 2. If ROS damage membranes and damaged membranes lead to ROS production then a feedforward loop can occur. Stresses that make ROS (e.g., ozone) and stresses that damage membranes (e.g., heat) will activate the feedforward cycle and lead to H₂O₂ accumulation and eventually cell death. Isoprene could stop this feedforward cycle in either of two ways: (1) quenching the ROS, and (2) stabilizing membranes. If isoprene quenches ROS the products of the isoprene/ROS interaction need to be considered. One prominent product is methylvinyl ketone, which could be cytotoxic.²⁷ The results of Velikova et al.¹⁴ allow speculation that membrane stabilization is a major mechanism by which isoprene helps plants tolerate abiotic stress of many different forms.

Figure 2. Simplified diagram showing two possible effects of isoprene (in green). If there is a central cycle in which ROS leads to membrane damage and membrane damage provides a signal for production of ROS (in blue), this feedforward loop could be initiated by many different stresses (e.g., ozone or heat stress) and could also be stopped by several different mechanisms (e.g., membrane stabilization or quenching of ROS). Isoprene is thought to lead to both, and indeed membrane are strengthened¹⁴ and ROS are quenched (this work, Figure 1) in genetically modified IspS Arabidopsis producing isoprene and exposed to heat stress. However, because heat stress primarily impinges on membranes, the demonstration by Velikova et al.¹⁴ that significant membrane stabilization is conferred by isoprene allows speculation that quenching of ROS is a side effect, and does not account for the protective effects observed for isoprene in heat stressed plants.

Velikova V, Várkonyi Z, Szabó M, Maslenkova L, Nogues I, Kovács V, et al. Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques. Plant Physiol. 2011;157:905–16. doi: 10.1104/pp.111.182519.