Ingo Voss

Emerging concept for the role of photorespiration as an important part of abiotic stress response

When plants are exposed to stress, generation of reactive oxygen species (ROS) is often one of the first responses. In order to survive, cells attempt to down-regulate the production of ROS, while at the same time scavenging ROS. Photorespiration is now appreciated as an important part of stress responses in green tissues for preventing ROS accumulation. Photorespiratory reactions can dissipate excess reducing equivalents and energy either directly (using ATP, NAD(P)H and reduced ferredoxin) or indirectly (e.g., via alternative oxidase (AOX) and providing an internal CO2 pool). Photorespiration, however, is also a source of H2 O2 that is possibly involved in signal transduction, resulting in modulation of gene expression. We propose that photorespiration can assume a major role in the readjustment of redox homeostasis. Protection of photosynthesis from photoinhibition through photorespiration is well known. Photorespiration can mitigate oxidative stress under conditions of drought/water stress, salinity, low CO2 and chilling. Adjustments to even mild disturbances in redox status, caused by a deficiency in ascorbate, AOX or chloroplastic NADP-malate dehydrogenase, comprise increases in photorespiratory components such as catalase, P-protein of glycine decarboxylase complex (GDC) and glycine content. The accumulation of excess reducing equivalents or ROS in plant cells also affects mitochondria. Therefore, a strong interaction between the chloroplast redox status and photorespiration is not surprising, but highlights interesting properties evident in plant cells. We draw attention to the fact that a complex network of multiple and dynamic systems, including photorespiration, prevents oxidative damage while optimising photosynthesis. Further experiments are necessary to identify and validate the direct targets of redox signals among photorespiratory components.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Knockout of major leaf ferredoxin reveals new redox-regulatory adaptations in Arabidopsis thaliana.

Ferredoxins are the major distributors for electrons to the various acceptor systems in plastids. In green tissues, ferredoxins are reduced by photosynthetic electron flow in the light, while in heterotrophic tissues, nicotinamide adenine dinucleotide (reduced) (NADPH) generated in the oxidative pentose-phosphate pathway (OPP) is the reductant. We have used a Ds-T-DNA insertion line of Arabidopsis thaliana for the gene encoding the major leaf ferredoxin (Fd2, At1g60950) to create a situation of high electron pressure in the thylakoids. Although these plants (Fd2-KO) possess only the minor fraction of leaf Fd1 (At1g10960), they grow photoautotrophically on soil, but with a lower growth rate and less chlorophyll. The more oxidized conditions in the stroma due to the formation of reactive oxygen species are causing a re-adjustment of the redox state in these plants that helps them to survive even under high light. Redox homeostasis is achieved by regulation at both, the post-translational and the transcriptional level. Over-reduction of the electron transport chain leads to increased transcription of the malate-valve enzyme NADP-malate dehydrogenase (MDH), and the oxidized stroma leads to an increased transcription of the OPP enzyme glucose-6-P dehydrogenase. In isolated spinach chloroplasts, oxidized conditions give rise to a decreased activation state of NADP-MDH and an activation of glucose-6-P dehydrogenase even in the light. In Fd2-KO plants, NADPH-requiring antioxidant systems are upregulated. These adjustments must be caused by plastid signals, and they prevent oxidative damage under rather severe conditions.

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP+. In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90–95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP+. Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP+ photoreduction.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

N-Terminal Structure of Maize Ferredoxin:NADP + Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

Maize chloroplasts conducting either cyclic or linear electron flow vary in their ferredoxin:NADP+ reductase (FNR) composition. By comparing FNR crystal structures and introducing modified FNRs back into plants, we show that N-terminal structure determines recruitment to different thylakoid complexes. Furthermore, electron flow is analyzed in plants enriched in FNR at alternative locations. To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP+ reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

Chapter 8 Use of Transgenic Plants to Uncover Strategies for Maintenance of Redox Homeostasis During Photosynthesis

Abstract Plant cells encounter a spectacular variation in the supply and consumption of redox components, due to changes in photosynthesis caused by the environment. To prevent these huge fluxes from causing catastrophic oxidative damage, there is an extensive network of compensatory, buffering mechanisms. These must be integrated with signaling cascades in a greater redox network, to ensure that short-term responses are adequate and that, if buffering capacity is exceeded, there is a response at the transcript level. Transgenic approaches have been fundamental in identifying the interconnections between redox fluxes, buffering, and signaling networks. In this review we discuss how this has shaped current understanding, and how transgenics might be used in the future to unravel the complex network required for energy metabolism, redox homeostasis, autotrophic growth and development under changing conditions. There are obvious problems associated with describing a highly interconnected network in the linear format of a written review, but we attempt this by first describing how redox poise is maintained in electron transport chains, move on to buffering pathways throughout the cell, and finally describe the mechanisms that detect signals, leading to interpretation of these changes at the level of altered transcription.

Expression of the Minor Isoform Pea Ferredoxin in Tobacco Alters Photosynthetic Electron Partitioning and Enhances Cyclic Electron Flow

Summary: Characterization of ferredoxin overexpressor transplastomic tobacco plants provides new evidence about the different roles of this protein and the effect of this feature on the regulation of the photosynthetic electron flow to produce alternative electron partitioning between linear and cyclic flow. Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.

Rhizoremediation of Diesel-Contaminated Soil with Two Rapeseed Varieties and Petroleum degraders Reveals Different Responses of the Plant Defense Mechanisms

Plant-assisted bioremediation (rhizoremediation) stands out as a potential tool to inactivate or completely remove xenobiotics from the polluted environment. Therefore, it is of key importance to find an adequate combination of plant species and microorganisms that together enhance the clean-up process. To understand the response of plants upon bioaugmentation, the antioxidative and detoxification system was analyzed in high and low erucic acid rapeseed varieties (HEAR and LEAR, respectively), after 8 weeks of their treatment with petroleum degraders and 6000 mg diesel oil/kg dry soil. The oxidative stress was enhanced in LEAR being exposed to sole diesel oil, in comparison with HEAR. However, when LEAR plants were additionally inoculated with bacteria, suppression of total catalase (CAT) and ascorbate peroxidase (APX) activity were observed. Interestingly, glutathione transferase (GST) activity was found in these plants at a much higher level than in HEAR, which correlated with a more efficient diesel removal performed by LEAR in the polluted soil and upon bioaugmentation. A distinct profile of polycyclic aromatic hydrocarbons (PAH) was detected in leaves of these plants. Neither LEAR nor HEAR experienced any changes in the photosynthetic capacity upon diesel pollution and presence of petroleum degraders, which supports the usefulness of rhizoremediation with rapeseed.