Characterization of the regulatory and expression context of an alternative oxidase gene provides insights into cyanide-insensitive respiration during growth and development

Alternative oxidase (AOX) is encoded in small multigene families in plants. Functional analysis of the Arabidopsis (Arabidopsis thaliana) alternative oxidase 1c (AtAOX1c) promoter, an AOX gene not induced by oxidative stress, indicated that regulation of expression was complex, with the upstream promoter region containing positive and negative response regions. Comparison to the promoter region of soybean (Glycine max) alternative oxidase 2b (GmAOX2b), another AOX gene not induced by oxidative stress, revealed that they contained seven sequence elements in common. All elements were active in the promoter region of AtAOX1c in suspension cells and in leaf tissue from Columbia and mutant plants, where a mitochondrial protein import receptor was inactivated. Analysis of coexpressed and putatively coregulated genes, the latter defined as containing five or more sequence elements functional in AtAOX1c, indicated that AtAOX1c was coregulated with components involved with cell division and growth. Consistent with this analysis, we demonstrated that site II elements, previously shown to regulate the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were strong negative regulators of AtAOX1c expression. It was demonstrated that NDB4, a gene encoding an external NAD(P)H dehydrogenase, displayed strong coexpression with AtAOX1c. Overall, these results indicate that AtAOX1c is regulated by growth and developmental signals.     

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.     

Modified alternative oxidase expression results in different reactive oxygen species contents in Arabidopsis cell culture but not in whole plants

Alternative oxidase (AOX) transfers electrons from ubiquinone to oxygen in the respiratory chain of plant mitochondria. It is widely accepted that AOX functions as a mechanism decreasing the formation of reactive oxygen species (ROS) produced during respiratory electron transport. However, there are no experimental data to provide unambiguous proof of this hypothesis. We have studied growth characteristics, ROS content, and stress sensitivity in Arabidopsis transgenic lines with reduced or increased levels of AOX. We demonstrated that AOX-deficient plants grown in soil had an extended reproductive phase. Changes in AOX activity did not affect ROS content or stress sensitivity in the whole plants. However in the suspension culture, cells overexpressing AOX had significantly lower ROS content, whereas the AOX-deficient cells had higher ROS contents compared to the wild-type (WT) cells. Prooxidant treatment led to the increase in ROS content and to the reduction of viability more in the cells overexpressing AOX than in WT and AOX-deficient cells. Thus, we demonstrated that differences in the metabolism of whole plants and cultured cells might affect AOX functioning.     

The overexpression of an alternative oxidase gene triggers ozone sensitivity in tobacco plants

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinione pool to oxygen without energy conservation and prevents the formation of reactive oxygen species (ROS) when the ubiquinone pool is over-reduced. Thus, AOX may be involved in plant acclimation to a number of oxidative stresses. To test this hypothesis, we exposed wild-type (WT) Xanthi tobacco plants as well as Xanthi plants transformed with the Bright Yellow tobacco AOX1a cDNA with enhanced (SN21 and SN29), and decreased (SN10) AOX capacity to an acute ozone (O3) fumigation. As a result of 5 h of O3 exposition (250 nL L(-1)), SN21 and SN29 plants surprisingly showed localized leaf damage, whereas SN10, similarly to WT plants, was undamaged. In keeping with this observation, WT and SN21 plants differed in their response to O3)for the expression profiles of catalase 1 (CAT1), catalase 2 (CAT2), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) genes, and for the activity of these antioxidant enzymes, which were induced in WT. Concomitantly, although ozone induced H2O2 accumulation in WT and in all transgenic lines, only in transgenics with high AOX capacity the H2O2 level in the post-fumigation period was high. The alternative pathway of WT plants was strongly stimulated by O3, whereas in SN21 plants, the respiratory capacity was always high across the treatment. The present results show that, far from exerting a protective role, the overexpression of AOX triggers an increased O3 sensitivity in tobacco plants. We hypothesize that the AOX overexpression results in a decrease of mitochondrial ROS level that in turn alters the defensive mitochondrial to nucleus signalling pathway that activates ROS scavenging systems.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Alternative Oxidase Expression in the Mouse Enables Bypassing Cytochrome c Oxidase Blockade and Limits Mitochondrial ROS Overproduction

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.     

Overexpression of wheat alternative oxidase gene Waox1a alters respiration capacity and response to reactive oxygen species under low temperature in transgenic Arabidopsis

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.     

In vivo cytochrome and alternative pathway respiration in leaves of Arabidopsis thaliana plants with altered alternative oxidase under different light conditions

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition.     

Alternative oxidase functions in the excess excitation energy-induced resistance to pathogen infection

Although excess excitation energy (EEE) can damage the photosynthetic apparatus and deregulate many cellular processes, some studies have reported that EEE can be used by plants to optimize the resistance to pathogen infection. Here, we investigated whether the EEE-induced resistance to pathogen infection might be mediated by mitochondrial alternative oxidase (AOX). The present work showed that exposure of Arabidopsis (Arabidopsis thaliana) leaves to short-term excess light or treatment of Arabidopsis leaves with the electron transport inhibitor, DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), restricted the growth of virulent biotrophic bacteria, Pseudomonas syringae pv tomato (Pst) DC3000. Exposure to short-term excess light or DBMIB treatment also enhanced the level of the capacity of the cyanide-resistant respiration of the infected leaves. The lack of AOX1a gene in the AtAOX1a anti-sense line attenuated the excess light- or DBMIB-induced resistance toward Pst DC3000. These results indicate that AOX may play a role in regulating the EEE-induced resistance to virulent pathogen infection.     

Overexpression of Alternative Oxidase Gene Confers Aluminum Tolerance by Altering the Respiratory Capacity and the Response to Oxidative Stress in Tobacco Cells

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.     

Alterations in the mitochondrial alternative NAD(P)H Dehydrogenase NDB4 lead to changes in mitochondrial electron transport chain composition, plant growth and response to oxidative stress

The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to an alternative oxidase (AOX). T-DNA and RNA interference (RNAi) were used to reduce gene expression to characterize the external NAD(P)H dehydrogenase NDB4 in Arabidopsis. The ndb4 lines showed different levels of suppression of NDB4 protein, leading to increases in NBD2 and AOX1a mRNA and protein levels in all lines. These changes were associated with lower reactive oxygen species formation and an altered phenotype, including changes in growth rate, root : shoot ratios and leaf area. The general growth pattern for the ndb4 mutants was decreased leaf area early in development (6-15 d) followed by a prompt subsequent increase in leaf area that exceeded the leaf area of the wild type by maturity (the 10-12 rosette stage). This pattern was most evident for the RNAi lines that had increased mitochondrial electron transport capacity. The RNAi lines also exhibited better tolerance to salinity stress, with better growth rates and lower shoot Na? content compared with controls when grown under saline conditions. We hypothesize that these differences reflect the enhanced expression of NDB2 and AOX in the ndb4 mutant plants.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex

Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.     

Harpin inactivates mitochondria in Arabidopsis suspension cells

Harpin is a well-known proteinaceous bacterial elicitor that can induce an oxidative burst and programmed cell death in various host plants. Given the demonstrated roles of mitochondria in animal apoptosis, we investigated the effect of harpin from Pseudomonas syringae on mitochondrial functions in Arabidopsis suspension cells in detail. Fluorescence microscopy in conjunction with double-staining for reactive oxygen species (ROS) and mitochondria suggested co-localization of mitochondria and ROS generation. Plant defense responses or cell death after pathogen attack have been suggested to be regulated by the concerted action of ROS and nitric oxide (NO). However, although Arabidopsis cells respond to harpin treatment with NO generation, time course analyses suggest that NO generation is not involved in initial responses but, rather, is a consequence of cellular decay. Among the fast responses we observed was a decrease of the mitochondrial membrane potential deltapsim, and, possibly as a direct consequence, of ATP production. Furthermore, treatment of Arabidopsis cells with harpin protein induced a rapid cytochrome C release from mitochondria into the cytosol, which is regarded as a hallmark of programmed cell death or apoptosis. Northern and DNA array analyses showed strong induction of protecting or scavenging systems such as alternative oxidase and small heat shock proteins, components that are known to be associated with cellular stress responses. In sum, the presented data suggest that harpin inactivates mitochondria in Arabidopsis cells.     

Distinct responses of the mitochondrial respiratory chain to long- and short-term high-light environments in Arabidopsis thaliana

In order to ensure the cooperative function with the photosynthetic system, the mitochondrial respiratory chain needs to flexibly acclimate to a fluctuating light environment. The non-phosphorylating alternative oxidase (AOX) is a notable respiratory component that may support a cellular redox homeostasis under high-light (HL) conditions. Here we report the distinct acclimatory manner of the respiratory chain to long- and short-term HL conditions and the crucial function of AOX in Arabidopsis thaliana leaves. Plants grown under HL conditions (HL plants) possessed a larger ubiquinone (UQ) pool and a higher amount of cytochrome c oxidase than plants grown under low light conditions (LL plants). These responses in HL plants may be functional for efficient ATP production and sustain the fast plant growth. When LL plants were exposed to short-term HL stress (sHL), the UQ reduction level was transiently elevated. In the wild-type plant, the UQ pool was re-oxidized concomitantly with an up-regulation of AOX. On the other hand, the UQ reduction level of the AOX-deficient aox1a mutant remained high. Furthermore, the plastoquinone pool was also more reduced in the aox1a mutant under such conditions. These results suggest that AOX plays an important role in rapid acclimation of the respiratory chain to sHL, which may support efficient photosynthetic performance.     

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the ¹⁸O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.     

Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves

The effects of growth irradiance and respiration on ascorbic acid (AA) synthesis and accumulation were studied in the leaves of wild-type and transformed Arabidopsis thaliana with modified amounts of the mitochondrial alternative oxidase (AOX) protein. Plants were grown under low (LL; 50 micromol photons m(-2) s(-1)), intermediate (IL; 100 micromol photons m(-2) s(-1)), or high (HL; 250 micromol photons m(-2) s(-1)) light. Increasing growth irradiance progressively elevated leaf AA content and hence the values of dark-induced disappearance of leaf AA, which were 11, 55, and 89 nmol AA lost g(-1) fresh weight h(-1), from LL-, IL-, and HL-grown leaves, respectively. When HL leaves were supplied with L-galactone-1,4-lactone (L-GalL; the precursor of AA), they accumulated twice as much AA and had double the maximal L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activities of LL leaves. Growth under HL enhanced dehydroascorbate reductase and monodehydroascorbate reductase activities. Leaf respiration rates were highest in the HL leaves, which also had higher amounts of cytochrome c and cytochrome c oxidase (CCO) activities, as well as enhanced capacity of the AOX and CCO electron transport pathways. Leaves of the AOX-overexpressing lines accumulated more AA than wild-type or antisense leaves, particularly at HL. Intact mitochondria from AOX-overexpressing lines had higher AA synthesis capacities than those from the wild-type or antisense lines even though they had similar L-GalLDH activities. AOX antisense lines had more cytochrome c protein than wild-type or AOX-overexpressing lines. It is concluded that regardless of limitations on L-GalL synthesis by regulation of early steps in the AA synthesis pathway, the regulation of L-GalLDH activity via the interaction of light and respiratory controls is a crucial determinant of the overall ability of leaves to produce and accumulate AA.     

Response of mitochondria to light intensity in the leaves of sun and shade species

The present authors have shown previously that both respiration rates and in vivo activities of the alternative oxidase (AOX) of leaves of Alocasia odora, a shade species, are lower than those in sun species, thereby optimizing energy production under limited light conditions (Noguchi et al., Australian Journal of Plant Physiology 28, 27–35, 2001). In the present study, mitochondria isolated from A. odora leaves were examined in order to investigate the biochemical basis for the differences in respiratory parameters. Alocasia odora and spinach plants were cultivated under both high and low light intensities, mitochondria were isolated from their leaves, and their respiratory properties compared. Mitochondrial content of leaf extracts from the two species was estimated using fumarase activities and antibody detection of porin (the voltage-dependent anion channel of the outer mitochondrial membrane). On a mitochondrial protein basis, spinach leaves showed higher capacities of the cytochrome pathway and cytochrome c oxidase (COX) than A. odora leaves. However, on a mitochondrial protein basis, A. odora showed higher capacities of AOX, which had a high affinity for ubiquinone when activated by pyruvate. Alocasia odora also had larger amounts of mitochondrial protein per leaf dry weight, even under severely shaded conditions, than spinach. Lower growth light intensity led to lower activities of most pathways and proteins tested in both species, especially glycine-dependent oxygen uptake. In the low light environment, most of the AOX protein in A. odora leaves was in its inactive, oxidized dimer form, but was converted to its reduced active form when plants were grown under high light. This shift may prevent over-reduction of the respiratory chain under photo-oxidative conditions.