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The degree of unsaturation of plant lipids is high, making them sensitive to oxidation. They thus constitute primary targets of reactive oxygen species and oxidative stress. Moreover, the hydroperoxides generated during lipid peroxidation decompose in a variety of secondary products which can propagate oxidative stress or trigger signaling mechanisms. Both primary and secondary products of lipid oxidation are helpful markers of oxidative stress in plants. This chapter describes a number of methods that have been developed to measure those biomarkers and signals, with special emphasis on the monitoring of photooxidative stress. Depending on their characteristics, those lipid markers provide information not only on the oxidation status of plant tissues but also on the origin of lipid peroxidation, the localization of the damage, or the type of reactive oxygen species involved., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plastoquinone-9 is known as a photosynthetic electron carrier to which has also been attributed a role in the regulation of gene expression and enzyme activities via its redox state. Here, we show that it acts also as an antioxidant in plant leaves, playing a central photoprotective role. When Arabidopsis plants were suddenly exposed to excess light energy, a rapid consumption of plastoquinone-9 occurred, followed by a progressive increase in concentration during the acclimation phase. By overexpressing the plastoquinone-9 biosynthesis gene SPS1 (solanesyl diphosphate synthase 1) in Arabidopsis, we succeeded in generating plants that specifically accumulate plastoquinone-9 and its derivative plastochromanol-8. The SPS1-overexpressing lines were much more resistant to photooxidative stress than the wild type, showing marked decreases in leaf bleaching, lipid peroxidation and PSII photoinhibition under excess light. Comparison of the SPS1 overexpressors with other prenyl quinone mutants indicated that the enhanced phototolerance of the former plants is directly related to their increased capacities for plastoquinone-9 biosynthesis.

Carotenoids play a crucial protective role in photosynthetic organisms as quenchers of singlet oxygen ((1)O(2)). This function occurs either via a physical mechanism involving thermal energy dissipation or via a chemical mechanism involving direct oxidation of the carotenoid molecule. The latter mechanism can produce a variety of aldehydic or ketonic cleavage products containing a reactive carbonyl group. One such molecule, the volatile β-carotene derivative β-cyclocitral, triggers changes in the expression of (1)O(2)-responsive genes and leads to an enhancement of photooxidative stress tolerance. Thus, besides their well-known functions in light harvesting and photoprotection, carotenoids can also play a role through their nonenzymic oxidation in the sensing and signalling of reactive oxygen species and photooxidative stress in photosynthetic organisms. Enzymic carotenoid oxidation does not seem to play a significant role in this phenomenon. Elucidation of the carotenoid-mediated (1)O(2) signalling pathway could provide new targets for improving photooxidative stress tolerance of plants.

The cellular redox balance seems an important modulator under heavy metal stress. While for other heavy metals these processes are well studied, oxidative stress related responses are also known to be triggered under uranium stress but information remains limited. This study aimed to further unravel the mechanisms by which plants respond to uranium stress. Seventeen-day-old Arabidopsis thaliana seedlings, grown on a modified Hoagland solution under controlled conditions, were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 1, 3 and 7 days. While in Part I of this study oxidative stress related responses in the roots were discussed, this second Part II discusses oxidative stress related responses in the leaves and general conclusions drawn from the results of the roots and the leaves will be presented. As several responses were already visible following 1 day exposure, when uranium concentrations in the leaves were negligible, a root-to-shoot signaling system was suggested in which plastids could be important sensing sites. While lipid peroxidation, based on the amount of thiobarbituric acid reactive compounds, was observed after exposure to 100 μM uranium, affecting membrane structure and function, a transient concentration dependent response pattern was visible for lipoxygenase initiated lipid peroxidation. This transient character of uranium stress responses in leaves was emphasized by results of lipoxygenase (LOX2) and antioxidative enzyme transcript levels, enzyme capacities and glutathione concentrations both in time as with concentration. The ascorbate redox balance seemed an important modulator of uranium stress responses in the leaves as in addition to the previous transient responses, the total ascorbate concentration and ascorbate/dehydroascorbate redox balance increased in a concentration and time dependent manner. This could represent either a slow transient response or a stable increase with regard to plant acclimation to uranium stress., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (1O2). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species., Results: The pdx1.3 mutation affects the vitamin B6 biosynthesis enzyme, pyridoxal synthase (PDX1), and leads to a reduction of the vitamin B6 concentration in Arabidopsis thaliana leaves. Although leaves of the pdx1.3 Arabidopsis mutant contained less chlorophyll than wild-type leaves, we found that vitamin B6 deficiency did not significantly impact photosynthetic performance or shoot and root growth. Chlorophyll loss was associated with an increase in the chlorophyll a/b ratio and a selective decrease in the abundance of several PSII antenna proteins (Lhcb1/2, Lhcb6). These changes were strongly dependent on light intensity, with high light amplifying the difference between pdx1.3 and the wild type. When leaf discs were exposed to exogenous 1O2, lipid peroxidation in pdx1.3 was increased relative to the wild type; this effect was not observed with superoxide or hydrogen peroxide. When leaf discs or whole plants were exposed to excess light energy, 1O2-mediated lipid peroxidation was enhanced in leaves of the pdx1.3 mutant relative to the wild type. High light also caused an increased level of 1O2 in vitamin B6-deficient leaves. Combining the pdx1.3 mutation with mutations affecting the level of 'classical' quenchers of 1O2 (zeaxanthin, tocopherols) resulted in a highly photosensitive phenotype., Conclusion: This study demonstrates that vitamin B6 has a function in the in vivo antioxidant defense of plants. Thus, the antioxidant activity of vitamin B6 inferred from in vitro studies is confirmed in planta. Together with the finding that chloroplasts contain vitamin B6 compounds, the data show that vitamin B6 functions as a photoprotector that limits 1O2 accumulation in high light and prevents 1O2-mediated oxidative damage.

Lipocalins are small ligand-binding proteins with a simple tertiary structure that gives them the ability to bind small, generally hydrophobic, molecules. Recent studies have shown that animal lipocalins play important roles in the regulation of developmental processes and are involved in tolerance to oxidative stress. Plants also possess various types of lipocalins, and bioinformatics analyses have predicted that some lipocalin members may be present in the chloroplast. Here we report the functional characterization of the Arabidopsis thaliana chloroplastic lipocalin AtCHL. Cellular fractionation showed that AtCHL is a thylakoid lumenal protein. Drought, high light, paraquat and abscisic acid treatments induce AtCHL transcript and protein accumulation. Under normal growth conditions, knockout (KO) and over-expressing (OEX) lines do not differ from wild-type plants in terms of phenotype and photosynthetic performance. However, KO plants, which do not accumulate AtCHL, show more damage upon photo-oxidative stress induced by drought, high light or paraquat. In contrast, a high level of AtCHL allows OEX plants to cope better with these stress conditions. When exposed to excess light, KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in OEX plants. The increased lipid peroxidation in KO plants is mediated by singlet oxygen and is not correlated with photo-inhibition of the photosystems. This work provides evidence suggesting that AtCHL is involved in the protection of thylakoidal membrane lipids against reactive oxygen species, especially singlet oxygen, produced in excess light.

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are necessary for the production of a variety of secondary metabolites, such as siderophores involved in iron acquisition. In response to iron limitation, the cyanobacterium Anabaena sp. strain PCC 7120 synthesizes several siderophores. The chromosome of this organism contains a large gene cluster of 76 kb with 24 open-reading frames from all2658 to all2635, including those that encode seven NRPSs and two PKSs. The function of this gene cluster was unknown, and one possibility could be the synthesis of siderophores. These genes were indeed activated under conditions of iron limitation. One mutant, MDelta41-49, bearing a large deletion of 43.4 kb in this gene cluster, synthesized considerably less siderophores and contained less iron as compared with the wild type. Its growth rate was similar to the wild type in the presence of iron, but was reduced when iron became limiting. Two other mutants, MDelta44-45 and MDelta47-49, lacking either all2644 and all2645, or all2647, all2648 and all2649 respectively, produced more siderophores than MDelta41-49, but less than the wild type. These genes were also activated under oxidative stress conditions to which MDelta41-49 was highly sensitive, consistent with the importance of iron in oxidative stress response. We propose that this gene cluster is involved in the synthesis of siderophores in Anabaena sp. PCC 7120 and plays an important role in defence against oxidative stress.

Reactive oxygen species act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-based quantitative method that allowed us to discriminate between free radical (type I)- and singlet oxygen ((1)O(2); type II)-mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method, we observed that in non-photosynthesizing Arabidopsis (Arabidopsis thaliana) tissues, nonenzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However, in leaf tissues under optimal growth conditions, (1)O(2) was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring (1)O(2) production, photooxidative stress led to a dramatic increase of (1)O(2) (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in (1)O(2)-dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, (1)O(2) plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.

The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the DeltapsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase (katG) and iron superoxide dismutase (sodB) was upregulated in the DeltapsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in DeltapsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the DeltapsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the DeltapsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.

Arabidopsis (Arabidopsis thaliana) plants were grown in a hydroponic culture system for 7 to 14 d in the absence or presence of 75 microM Cd or 75 microM Cu. The Cu treatment resulted in visual leaf symptoms, together with anthocyanin accumulation and loss of turgor. Pronounced lipid peroxidation, which was detected by autoluminescence imaging and malondialdehyde titration, was observed in Cu-treated leaves. The Cd treatment also resulted in loss of leaf pigments but lipid peroxidation and oxidative stress were less pronounced than in the leaves exposed to Cu. Analysis of low-molecular-weight chloroplast and cytosolic antioxidants (ascorbate, glutathione, tocopherols, carotenoids) and antioxidant enzymes (thiol-based reductases and peroxidases) revealed relatively few responses to metal exposure. However, there was a marked increase in vitamin E (alpha-tocopherol) in response to Cd and Cu treatments. Ascorbate increased significantly in Cu-exposed leaves. Other antioxidants either remained stable or decreased in response to metal stress. Transcripts encoding enzymes of the vitamin E biosynthetic pathway were increased in response to metal exposure. In particular, VTE2 mRNA was enhanced in Cu- and Cd-treated plants, while VTE5 and hydroxylpyruvate dioxygenase (HPPD) mRNAs were only up-regulated in Cd-treated plants. Consistent increases in HPPD transcripts and protein were observed. The vitamin E-deficient (vte1) mutant exhibited an enhanced sensitivity towards both metals relative to the wild-type (WT) control. Unlike the vte1 mutants, which showed enhanced lipid peroxidation and oxidative stress in the presence of Cu or Cd, the ascorbate-deficient (vtc2) mutant showed WT responses to metal exposure. Taken together, these results demonstrate that vitamin E plays a crucial role in the tolerance of Arabidopsis to oxidative stress induced by heavy metals such as Cu and Cd.

Living organisms spontaneously emit faint light, and this autoluminescence is stimulated in response to many stresses. This phenomenon is attributable to the endogenous production of excited states during oxidative reactions, particularly during peroxidation of lipids, which generates light-emitting molecules such as triplet carbonyls and singlet oxygen. Using highly sensitive cameras, it is now possible to remotely image spontaneous luminescence with a good spatial resolution, providing a new non-invasive tool for mapping oxidative stress and lipid peroxidation in plants.

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

Vitamin E is considered a major antioxidant in biomembranes, but little evidence exists for this function in plants under photooxidative stress. Leaf discs of two vitamin E mutants, a tocopherol cyclase mutant (vte1) and a homogentisate phytyl transferase mutant (vte2), were exposed to high light stress at low temperature, which resulted in bleaching and lipid photodestruction. However, this was not observed in whole plants exposed to long-term high light stress, unless the stress conditions were extreme (very low temperature and very high light), suggesting compensatory mechanisms for vitamin E deficiency under physiological conditions. We identified two such mechanisms: nonphotochemical energy dissipation (NPQ) in photosystem II (PSII) and synthesis of zeaxanthin. Inhibition of NPQ in the double mutant vte1 npq4 led to a marked photoinhibition of PSII, suggesting protection of PSII by tocopherols. vte1 plants accumulated more zeaxanthin in high light than the wild type, and inhibiting zeaxanthin synthesis in the vte1 npq1 double mutant resulted in PSII photoinhibition accompanied by extensive oxidation of lipids and pigments. The single mutants npq1, npq4, vte2, and vte1 showed little sensitivity to the stress treatments. We conclude that, in cooperation with the xanthophyll cycle, vitamin E fulfills at least two different functions in chloroplasts at the two major sites of singlet oxygen production: preserving PSII from photoinactivation and protecting membrane lipids from photooxidation.

We establish here that iron deficiency causes oxidative stress in the cyanobacterium Anabaena sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.

The products of the isiAB operon are a chlorophyll antenna protein (IsiA) and flavodoxin (IsiB), which accumulate in cyanobacteria grown under iron starvation conditions. Here we show that strong light triggers de-repression of isiAB transcription and leads to IsiA and flavodoxin accumulation under iron replete conditions. Genetic deletion of isiAB resulted in a photosensitive phenotype, with accumulation of reactive oxygen species and cell bleaching in high light, while the flavodoxin-deficient isiB null mutant expressing isiA was phototolerant. We conclude that IsiA protects cyanobacteria from photooxidative stress. IsiA is the first example of a chlorophyll antenna protein outside the extended LHC family that is induced transiently by high light and that fulfills a photoprotective role.

When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids. In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light. Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions. In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides. Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast. Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis. From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide. The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes.

Peroxidation of polyunsaturated fatty acids is one of the main events triggered by oxidative stress in cells. Some lipid peroxidation products are light-emitting species, and their luminescence can be used as an internal marker of oxidative stress. However, this spontaneous chemiluminescence is weak and difficult to measure. Recent studies have shown that an alternative approach that involves measuring thermoluminescence bands at high temperature (in the range 80-150 degrees C) is a simple way of detecting and quantifying lipid peroxidative damage and oxidative stress in plants.

Studies of the singlet oxygen (¹O2)-overproducing flu and chlorina1 (ch1) mutants of Arabidopsis (Arabidopsis thaliana) have shown that ¹O2-induced changes in gene expression can lead to either programmed cell death (PCD) or acclimation. A transcriptomic analysis of the ch1 mutant has allowed the identification of genes whose expression is specifically affected by each phenomenon. One such gene is OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) encoding an AGC kinase that was noticeably induced by excess light energy and ¹O2 stress conditions leading to cell death. Photo-induced oxidative damage and cell death were drastically reduced in the OXI1 null mutant (oxi1) and in the double mutant ch1*oxi1 compared with the wild type and the ch1 single mutant, respectively. This occurred without any changes in the production rate of ¹O2 but was cancelled by exogenous applications of the phytohormone jasmonate. OXI1-mediated ¹O2 signaling appeared to operate through a different pathway from the previously characterized OXI1-dependent response to pathogens and H2O2 and was found to be independent of the EXECUTER proteins. In high-light-stressed plants, the oxi1 mutation was associated with reduced jasmonate levels and with the up-regulation of genes encoding negative regulators of jasmonate signaling and PCD. Our results show that OXI1 is a new regulator of ¹O2-induced PCD, likely acting upstream of jasmonate. [ABSTRACT FROM AUTHOR]

The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis. Singlet oxygen (1O2) is a highly reactive species to which has been attributed a major destructive role during the execution of ROS-induced cell death in photosynthetic tissues exposed to excess light. The study of the specific biological activity of 1O2 in plants has been hindered by its high reactivity and short lifetime, the concurrent production of other ROS under photooxidative stress, and limited in vivo detection methods. However, during the last 15 years, the isolation and characterization of two 1O2-overproducing mutants in Arabidopsis thaliana, flu and ch1, has allowed the identification of genetically controlled 1O2 cell death pathways and a 1O2 acclimation pathway that are triggered at sub-cytotoxic concentrations of 1O2. The study of flu has revealed the control of cell death by the plastid proteins EXECUTER (EX)1 and EX2. In ch1, oxidized derivatives of β-carotene, such as β-cyclocitral and dihydroactinidiolide, have been identified as important upstream messengers in the 1O2 signaling pathway that leads to stress acclimation. In both the flu and ch1 mutants, phytohormones act as important promoters or inhibitors of cell death. In particular, jasmonate has emerged as a key player in the decision between acclimation and cell death in response to 1O2. Although the flu and ch1 mutants show many similarities, especially regarding their gene expression profiles, key differences, such as EXECUTER-independent cell death in ch1, have also been observed and will need further investigation to be fully understood. [ABSTRACT FROM AUTHOR]

A psaFJ-null mutant of Synechocystis sp. strain PCC 6803 was characterised. As opposed to similar mutants in chloroplasts of green algae, electron transfer from plastocyanin to photosystem 1 was not affected. Instead, a restraint in full chain photosynthetic electron transfer was correlated to malfunction of photosystem 1 at its stromal side. Our hypothesis is that absence of PsaF causes oxidative stress, which triggers the induction of the ‘iron stress inducible’ operon isiAB. Products are the IsiA chlorophyll-binding protein (CP43′) and the isiB gene product flavodoxin. Supporting evidence was obtained by similar isiAB induction in wild type cells artificially exposed to oxidative stress. [Copyright &y& Elsevier]

Abstract: Barley displays a great genetic diversity, constituting a valuable source to delineate the responses of contrasted genotypes to environmental constraints. Here, we investigated the level of oxidative stress and the participation of antioxidant systems in two barley genotypes: Express, a variety known to be sensitive to drought, and Saïda, an Algerian landrace selected for its tolerance to water deficit. Soil-grown 15-day-old plants were subjected to water deficit for 8 days and then rewatered. We observed that upon water stress Express exhibits compared to Saïda accelerated wilting and a higher level of oxidative stress evaluated by HPLC measurements of lipid peroxidation and by imaging techniques. In parallel, Express plants also display lower levels of catalase and superoxide dismutase activity. No great difference was observed regarding peroxiredoxins and methionine sulfoxide reductases, enzymes detoxifying peroxides and repairing oxidized proteins, respectively. In contrast, upon water stress and recovery, much higher contents and oxidation ratios of glutathione and ascorbate were measured in Express compared to Saïda. Express also shows during water deficit greater increases in the pools of lipophilic antioxidants like xantophyll carotenoids and α-tocopherol. Altogether, these data show that the differential behavior of the two genotypes involves distinct responses regarding antioxidant mechanisms. Indeed, the drought sensitivity of Express compared with Saïda is associated with oxidative damage and a lower enzymatic ROS-scavenging capacity, but in parallel with a much stronger enhancement of most mechanisms involving low-molecular weight antioxidant compounds. [Copyright &y& Elsevier]