The gene expression profiles of mitochondrial respiratory components in Arabidopsis plants with differing amounts of ALTERNATIVE OXIDASE1a under high intensity light

ABSTRACT

We compared the expression of mitochondrial alternative oxidase (AOX) and other non-phosphorylating respiratory components (NPhPs) in wild type and AOX1a transgenic Arabidopsis thaliana following short-term transfer of plants to higher irradiance conditions to gain more insight into the mechanisms of AOX functioning under light. The AOX1a overexpressing line (XX-2) showed the highest amount of AOX1a transcripts and AOX1A synthesis during the entire experiment, and many NPhPs genes were down-regulated after 6–8 h under the higher light conditions. Antisense AS-12 plants displayed a compensatory effect, typically after 8 h of exposure to higher irradiance, by up-regulating their expression of the majority of genes encoding AOX and other respiratory components. In addition, AS-12 plants displayed ‘overcompensation effects’ prior to their transfer to high light conditions, i.e., they showed a higher expression level of certain genes. As a result, the ROS content in AS-12, as in XX-2, was consistently lower than in the wild type. All NPhPs genes share, in common with AOX1a, light- and stress-related cis-acting regulatory elements (CAREs) in their promoters. However, the expression of respiratory genes does not always depend on the level of AOX1a expression. This suggests the presence of multiple combinations of signaling pathways in gene induction. Based on our results, we outline possible directions for future research.

It is known that mitochondrial respiration under light optimizes photosynthesis and maintains the cellular redox balance.^1–4 In many respects, it is connected with the functioning of the mitochondrial electron transport chain (mETC), which is in contrast to mammalian mETC as it consists not only of a phosphorylating cytochrome pathway (CP) but also of several non-phosphorylating pathways (NPhPs). These pathways are not coupled with a transmembrane proton producing an electrochemical gradient. As a result, ATP synthesis is bypassed. Additional enzymes involved include the rotenone-insensitive internal and external type II NAD(P)H dehydrogenases (NDs) ,⁵ uncoupling proteins (UCP) which uncouple electron transport to ADP phosphorylation ,⁶ and another protein which has a strong effect on cell energy, known as the terminal cyanide-insensitive alternative oxidase (AOX).^7,8 AOX transfers electrons directly from the ubiquinone pool to oxygen and produces water. Consequently, the AOX pathway bypasses proton-pumping Complexes III and IV and reduces the energy yield of respiration by at least two thirds. These energy-dissipating systems (AOX, NDs, and UCP) allow for the fine-tuning of mitochondrial membrane potential and the alleviation of ROS production. Among them, the alternative oxidase pathway (AP) plays a crucial role in the photosynthesizing cell.^1,9–13 AOX is involved in the dissipation of excess reductants exported from chloroplasts, thereby ‘unloading’ photosynthetic ETC and maintaining cell redox homeostasis.

AOX and other enzymes of NPhPs are encoded by nuclear genes. AOX gene expression is thought to be directly (anterograde) light induced and mediated through photoreceptor control.^12,14,15 Photosensitive cis-acting regulatory elements (CAREs) that regulate gene expression are found to be evenly distributed over the 1.0 kb AtAOX1a promoter.^14,16 The G-box sequence, the known CARE in the promoters of many light-regulated genes, has been also identified in the promoter region of AOX1a in Arabidopsis.¹⁷ The expression of AOX is also regulated by retrograde signals induced by perturbed mitochondrial function. The mitochondrial retrograde regulation (MRR) region (a 93 bp portion) in the promoter of AOX1a in Arabidopsis was found to be essential for gene induction by the mETC and TCA cycle inhibitors.¹⁸ Afterward, 10 distinct CAREs, associated with stress- and light responses, in the AtAOX1a promoter were discovered.¹⁷ ROS production and changes in the TCA cycle and TCA acids under high light conditions may trigger the activation of CAREs in the MRR region via interactions with appropriate transcription factors.^18,19 Changes in the redox state in the photosynthetic electron transport chain (for example, at the level of the plastoquinone pool) may also act as a signal transduced to the nucleus and trigger gene expression, including components of mitochondrial respiration.²⁰ Close correlations between the plastoquinone redox state and the expression of AOX and chloroplast light-harvesting complex II (LHCB2) components under light- and drought-induced stress were recently shown.²¹ The signaling pathways involved in light induction of AOX gene expressions are still not fully understood.^15,20

The important role of AOX in supporting efficient photosynthetic performance has been confirmed in studies, which involved the manipulation of AOX gene expression. In Arabidopsis thaliana (L.) Heynh., AOX1a is the most stress-responsive gene, among all AOX genes, in terms of the variety of stress inducers it responds to and the magnitude of its response.^17,18,22 In AOX1a-knockout A. thaliana mutants (aox1a) or SHAM-treated wild type plants during greening under higher light (500 μmol m⁻² s⁻¹) conditions a reduced NADP⁺/NADPH ratio decreases plastidial protein import efficiency.²³ In addition, overexpression of AOX1a rescues the aox1a mutant, and induces chlorophyll accumulation and plastidial protein import. The decline in the maximum quantum efficiency of photosystem II is significantly faster in the aox1a mutants than in the wild type.²⁴ A reduction in ZAT10 mRNA (a transcriptional repressor involved in abiotic stress responses) in aox1a A. thaliana plants has been shown to impair tolerance to ROS and HL conditions and to decrease the expression of antioxidant genes.²⁵ AOX1a-antisense plants suffer from more severe PSII photoinhibition after exposure to high light (800 μmol m⁻² s⁻¹) conditions than wild type (WT) or AOX1a-overexpressed plants.²⁶ This decreased ability of aox1a mutants to withstand oxidative stress can be explained by the fact that other AOX isoforms cannot fully compensate for the lack of AOX1A.^27,28 There are also studies where mutants that suppress or lack AOX display a compensatory effect by activating other NPhPs and ROS defense systems in response to stress.^{16,25,29–32} These contradictory findings may be explained by the nature, duration, and intensity of the stress treatment.

Strong alterations in the phenotype of AOX-transformed plants under stress, including HL conditions as well as early light induction of AOX expression ,^12,14,20,33 suggests that changes in the AOX amount will lead to compensatory changes in the expression of other respiratory components. It cannot be ruled out that AOX expression, as a component integrated into a wider transcriptional stress response, changes the redox status and affects the involvement of other defense metabolic pathways. In experiments on the de-etiolation of a wheat leaf, the expression of several NPhP genes lagged behind the expression of AOX1a.^12,34 This is consistent with the idea that AOX is not only a target but a regulator of stress responses.³⁵ It is also been proposed that AOX is a marker of genetic variation in cell reprogramming and yield stability.³⁶ However, the role of AOX in triggering and/or mediating the induction of other stress-response genes is not conclusive and is currently the subject of research.

Here, a wild type and two AOX1a-transformed A. thaliana lines (an overexpressing line and an antisense line with strongly decreased protein expression) were transferred to higher irradiance conditions to gain more insight into the mechanisms involved in the regulation of AOX under light and the role of AtAOX1a in the expression of other AOX and NPhPs genes. A search for common CAREs in the promoters of AOX1a and other studied genes was performed to detect possible interactions between genes at the transcriptional level.

Arabidopsis (A. thaliana) plants, the Col-0 ecotype (wild type, WT), and AtAOX1a antisense (AS-12) and overexpression (XX-2) lines (for further details, see³⁷) were grown at 22°C for 10 h under 90 μmol m⁻² s⁻¹ light conditions (using luminescent lamps TL-D 30 W and TL-D 30 W (Aquarelle, Philips, Amsterdam, the Netherlands) and 14 h of darkness. Seeds from the WT and the AOX-modified plants were obtained from the Nottingham Arabidopsis Stock Center (NASC, Nottingham University, UK), and stock numbers were N6707 and N6591 for AS-12 and XX-2, respectively (kindly presented by Dr. V. I. Tarasenko and Dr. O. I. Grabelnych from the Siberian Institute of Plant Physiology, Irkutsk, Russia).³⁸ Four-week-old plants were transferred to the higher irradiance (400 μmol m⁻² s⁻¹) treatment for 8 h. A value of 400 µmol m^–2 s^–1 was found to be the level of illumination in the upper part of the curvature region (the transition from the light-limited phase to the light-saturated plateau) on the light-response curve obtained for the net photosynthetic rate of the mature rosette leaf (Supplementary Fig. S1). Gene expression analysis was performed using Real-Time quantitative Reverse Transcription PCR (qRT-PCR) (for further details see:¹⁶). Primer pairs were designed using the Primer-BLAST online tool³⁹ to simultaneously amplify fragments of all known splice variants of each of the studied genes¹⁶ The expressions of the studied genes were normalized using the housekeeping genes AT2G28390 and AT4G34270.⁴⁰ For Western-blotting the combined leaf extracts from the wild type were separated on 12.5% gel and blotted on a nitrocellulose membrane using a wet transfer (25 mM Tris, 192 mM glycine, and 10% (v/v) methanol at pH 8.3).¹⁶ Filters were blocked (1.5 h) in 2% milk in TBST and incubated with polyclonal antibodies (Agrisera, Vännäs, Sweden) at a dilution factor of 1:1000. Anti-AOX1/2 (AS04 054), anti-COXII (AS04 052), and anti-VDAC1 (AS07 212) were incubated at a dilution factor of 1:5000. Polyclonal antibodies, anti-NDA1 (PHY 1037S), anti-NDA2 (PHY1038S), anti-NDB2 (PHY1555S), anti-UCP1 (PHY1574S) (PhytoAB Inc., San Francisco, California, USA), were incubated at a dilution factor of 1:1000. Secondary antibodies linked to horseradish peroxidase were then used and the signal was detected by chemiluminescence using Quantity One 1-D Analysis Software, Version 4.6.9. (Bio-Rad, California, USA). Superoxide (O₂⁻) and peroxide were spectrophotometrically determined according to Chaitanya abd Naithani⁴¹ and to Bellincampi et al.,⁴² respectively, and described in detail by Garmash et al.¹² Thiobarbituric acid reactive substance (TBARS) determinations were performed using the method described by Heath and Packer.⁴³

After the plants were transferred to higher light conditions, elevated expressions of four AOX genes, including AOX1a in the antisense line and three genes (AOX1a, AOX1c, and AOX2) in the WT, were detected. XX-2 plant samples were characterized by stable high levels of AOX1a expression, and some genes were even down-regulated (Figure 1A). AOX1b expressions were very low in all lines, although AOX1b is in tandem with AOX1a on chromosome 3 in A. thaliana. However, AOX1b expression is limited to flowers and floral buds.²² The antisense line had low and quite leaky expression of AOX1a, also, following the transfer to higher irradiance conditions. Interestingly, the highest AOX1d expression in the AS-12 line was evident 8 h after transfer of plants to HL conditions. In A. thaliana, AOX1d (along with AOX1a) is known to be highly responsive to stress, in contrast to other AOX genes.^22,27,46 In some cases stress-induced expressions of AOX1c and AOX2 have also been identified.^47,48

Figure 1.

Expression profiles of alternative oxidase genes (AOX1a-d and AOX2) (A) and genes encoding other non-phosphorylating respiratory components (B-D) in leaves of wild type (WT), XX-2, and AS-12 Arabidopsis thaliana plants grown in control (at 90 μmol m⁻² s⁻¹, i.e. 0 h) and in higher light conditions (at 400 μmol m⁻² s⁻¹, i.e. 2–8 h). The genes encoding internal NAD(P)H dehydrogenases (NDA1,2 and NDC1) (B), external NAD(P)H dehydrogenases (NDB1-4) (C), and uncoupling proteins (UCP1-5) (D) were examined. (E) A hierarchical cluster analysis was performed with the R-program package pvclast using the Ward method of clustering and absolute correlation values as distance.^44,45 Three plants were combined into one common sample to isolate total RNA and synthesize cDNA. Each data point represents three pooled samples (i.e. nine plants). Three RT-PCR reactions were performed on each pooled sample. The final number of expression values (nine) was used as n for statistical calculations. Significant differences between mean values in each gene analysis are indicated by different letters (ANOVA, Duncan’s test, P < 0.05)

Figure 1.

(continued)

In order to analyze a possible interaction between AOX genes at the transcriptional level, a study of a 1000 bp fragment belonging to the upstream regions of the transcription start sites (TSS) of all AtAOX genes was carried out by manually searching the NCBI (National Center of Biotechnology Information) Gene data bases and by using the Arabidopsis Gene Regulatory Information Server (Agris) (Figure 2). Results showed that all AtAOX genes have several light- and stress-regulated cis-acting regulatory elements (CAREs) in common (with the exception of AOX1b, which has only one CARE in common) with AOX1a. The presence of several G-box elements (CACGTG) in AOX1a, AOX2 promoters, and the co-expression of these genes indicate that their light induction is connected with the HY5-activation signal pathway. HY5 is a positive regulator of photomorphogenesis under broad-spectrum light conditions, suggesting that it acts downstream of phyto- and crypto-chromes (reviewed in⁴⁹). It encodes the G-box binding factor (GBF1), which is a basic leucine zipper transcription factor (TF) (bZIP) that binds to G-box and promotes many light-regulated genes including those related to photosynthesis.^50,51 The expressions of AOX1a, AOX1c, and AOX1d, which are enriched by CAREs such as the MYB4 binding site motif (BSM), SORLEP3, SORLIP2, and CCA1 BSM in their promoters, are possibly more dependent on perturbations in the photosynthetic electron transport chain under changing light conditions. CCA1 is known to bind to TFs in the MYB family, and is involved in the phytochrome-regulated expression of the LHCB gene, which encodes light-harvesting chlorophyll a/b proteins associated with photosystem II in Arabidopsis.^52,53 LHCB plays an important role in the photoprotection of PSII when plants are exposed to HL conditions.^52,54 MYB4, like other MYB TFs, is implicated in different plant stress responses.⁵⁵ SORLEP3 and SORLIP2 may be involved in mediating responses downstream of phyA, perhaps in combination with other elements under stress conditions including high light.⁵³ It can be concluded that the AOX1a promoter region contains light- and stress-responsive CAREs that occur in the promoters of other AOX genes. This suggests that other AOX genes may compensate for AOX1a suppression at the transcriptional level, at certain illumination levels. Contrarily, expression of AOX genes may be regulated by distinctive signaling pathways that converge to act on shared CAREs. This was indicated by the hierarchical clustering algorithm we used, which selected AOX genes from three different clusters (Figure 1E). However, much more research is needed to understand the regulation of AOX genes’ expression at the transcriptional level under light stress.

Figure 2.

Amounts of cis-acting regulatory elements (CAREs) in promoters of Arabidopsis thaliana genes encoding components of alternative and cytochrome respiratory pathways. Analysis of a 1000 bp fragment belonging to the upstream regions of all genes, made by manual search using the NCBI databases and known sequences of CAREs (MRE- and ACE-like boxes) as well as the analysis of promoters using the Agris database. Light-responsive CAREs: G-box (CACGTG), MRE-like box (ACCTA), ACE-like box (CACGTG), CCA1 binding site motive (BSM) (AA(A/C)AATCT), SORLIP2 (GGGCC), and SORLREP3 (TGTATATAT). Stress responsive CAREs: MYB4 BSM (A(A/C)C(A/T)A(A/C)C), and ABRE BSM ((C/G/T)ACGTG(G/T)(A/C))

We found that light induction of AOX gene expression was followed by an increase in the amount of different AOX proteins in the WT and transformant lines (Figure 3, Supplementary Fig. S2). Two bands on the AOX immunoblots at approximately 34 and 30 kDa, probably corresponding to different isoforms of the AOX1 reduced form, were detected (Figure 3). The presence of a second lower molecular band (30 kDa) in AOX Western-blot analysis has also been described in previous studies on isolated mitochondria from Arabidopsis plants.^56–58The AOX protein associated with the 34 kDa band was found in abundance in leaves from the XX-2 line. This is likely a product of AOX1a expression (AOX1A).³⁷ WT and AS-12 plants, before exposure to higher irradiance conditions, contained a very low amount of AOX1 (at the 34 kDa band). After exposure to higher irradiance, levels of the AOX1 protein at the 34 kDa band markedly increased in leaves from the WT and AS-12 plants. Since the amount of sense AOX1a can be present at up to 50% in AS-12 plants relative to the wild type ,³⁷ AOX (at the 34 kDa band) in samples of the antisense line under the higher light conditions was likely a product of AOX1a expression.

Figure 3.

Immunoblots of AOX (two bands around 34 and 30 kDa), internal NAD(P)H dehydrogenases (NDA1, and NDA2), external NAD(P)H dehydrogenases (NDB2), an uncoupling protein (UCP1), and mitochondrial voltage-dependent anion channel (VDAC1) proteins in whole-leaf tissue extracts from the wild type (Col-0) and two AOX1a-transformed lines grown in control (at 90 μmol m⁻² s⁻¹, i.e. 0 h) and in higher light conditions (at 400 μmol m⁻² s⁻¹, i.e. 2–8 h) (A). Fifteen micrograms of protein were loaded per lane, separated by an SDS-PAGE, immunoblotted, and visualized by chemiluminescence. Relative values of proteins levels, using VDAC1 as a reference, were obtained by dividing the densitometry of the appropriate protein and the VDAC1 bands (B). Blots were quantified by densitometry using Quantity One 4.6.9 software (Bio-Rad, USA). Data are presented as mean values ± SE of three replicates. Significant differences between mean values in each protein analysis are indicated by different letters (ANOVA, Kruskal-Wallis test, P < 0.05)

The AOX1A, AOX1B, AOX1C, and AOX1D precursor proteins deduced from the nucleotide sequences of AOX1a, AOX1b, AOX1c, and AOX1d are 354, 325, 329, and 318 amino acid residues, respectively (,⁵⁹ TAIR, AOX1D: AT1G32350.1). We cannot attribute the AOX isoform at the 30 kDa band to a specific gene and, thus, believe that the 30 kDa AOX isoform is a mixed product resulting from the expressions of AOX1b-d genes.

Stress-induced co-expression of AOX and genes related to other NPhPs in plants, including A. thaliana, has often been observed. This suggests that the presence of multiple members in the alternative respiratory components pathway provide flexibility and fine control in response to various stresses.^{12,16,20,60,61} There is evidence of AOX1a-dependent expression of genes of other NPhPs.²⁰ Contrarily, it has been shown that AOX1a expression is unaffected by the silencing of, for example, NDs genes.^62–64 With the incorporation of the AtNDB2 overexpression construct into the AtAOX1a-overexpressed background line XX1, the expression of AtNDB2 in the mitochondria has been found to strongly increase.⁶⁵ However, the amount of AOX protein was unchanged.⁶⁵ It has also been shown that other respiratory components may compensate for the absence of functional AOX1a ,^16,30,47 suggesting a modulating role for AOX1a in the functioning of the mETC, especially during oxidative challenges in response to stress.

Following transfer to higher irradiance conditions, gene expression in the NPhPs components was mainly correlated with the synthesis of the studied encoding proteins and depended on the level of AOX1a expression (Figure 1). In the AS-12 line, increases in the expressions of most NPhPs genes (NDA1,2, NDC1, NDB1,2,3, and UCP1,2,3,5) were observed. The relative transcript amounts of NDA2, NDB3, and UCP1 increased exclusively in AS-12, and NDA1 and UCP2 increased in both the AOX1a-transformed lines. This indicates that the expression of NPhPs genes is dependent on the level of AOX1a expression.

We analyzed the available information on expression patterns of the above listed genes and their co-expression with AOX1a under changing light conditions, as well as the common light- and stress-regulated CAREs in promoter regions of these genes. In the WT line, the gene expression and protein synthesis of one of the internal NDs, NDA1, were relatively high even before exposure to the higher light conditions, but then these decreased by the end of the experiment (8 h) (Figures 1 and 3). This may be attributed to a diurnal NDA1 expression pattern, which is at its highest expression level 1–2 h after the onset of day, followed by a steep decrease.^20,66,67 In the present study, plants were transferred to higher irradiance conditions 2 h after the onset of day. Thus, NDA1 expression and encoding protein synthesis may be fully induced before transfer due to diurnal changes. In AOX1a transformants, NDA1 expression and NDA1 synthesis were noticeably different compared to the WT line, as they were amplified under HL conditions. The highest numbers of the NDA1 promoter were observed in identical light-related CAREs related to the AOX1a promoter. In addition, AOX1a and NDA1 were combined in one close cluster (Figure 1E). Thus, the cooperation of NDA1 and AOX1a occurs at the mRNA level. It was demonstrated that NDA1 expression, as well as that of AOX1a, is maintained at a high level under methyl viologen treatment and high CO₂ levels under higher light conditions 400 μmol m⁻² s⁻¹).²⁰ This suggests that ROS generated from the photosynthetic system and the cellular carbon status respectively contribute to the regulation of both gene expression and their co-expression.²⁰ Our results indicate that the higher expression of NDA1 and the synthesis of NDA1 in the XX-2 line are related to the co-expression of genes (AOX1a and NDA1) and, in the AS-12 line, these genes interact in a compensatory manner.

NDA2 encodes an internal NADH dehydrogenase and is as involved as NDA1 in the oxidation of matrix NADH.⁵ We found that the level of expression of NDA2 was lower than that of NDA1 and increased gradually in all genotypes under HL conditions. NDA2 expression in A. thaliana has not been found to have distinct responses to HL and dark conditions.²⁰ In addition, in comparison to NDA1, the NDA2 promoter had lower numbers in identical CAREs that were related to the AOX1a promoter. Interestingly, NDA2 belongs to another cluster tribe within the same large cluster as AOX1a.

Co-expression of AtNDB2 with AtAOX1a in treatment responses has often been reported.^20,60 Both genes have identical functional stress-responsive CAREs, including I1, which is responsible for the MRR-dependent induction of their expression.^17,18 Based on our analysis, promoters of NDB2 had, like NDA1, a large number of factors in common, especially stress-related CAREs, with the AOX1a promoter. However, expressions of NDB2 increased in all lines (Figure 1C). The co-expression of AOX1a and NDB2 in illuminated A. thaliana leaves has not always been observed (due to variations in light regimes, CO₂ levels, or chemical treatments).²⁰ According to our cluster analysis results, NBD2 is in the same cluster as AOX1a, but in a different sub-cluster (like NDA2). This is consistent with the idea that different signaling pathways regulate the expression of AOX1a and NDB2.¹⁷ It has been suggested that perturbations in the photosynthetic electron transport chain may be involved in the alteration of NDB2 expression.²⁰

The expressions of UCP1 and UCP5 were similar to the observed patterns in NDB2 expression (Figure 1D). The expressions of these UCP genes have been shown to be highly induced under different stress conditions in A. thaliana, including HL.^6,20,30,47 However, among other UCPs, the promoters of UCP1 and UCP5 showed the lowest and the highest numbers of observations, respectively, in CAREs in relation to the AOX1a promoter. In addition, all UCP genes were combined in the same AOX1d cluster. These data together indicate that UCP genes are relatively independent at the level of AOX1a expression under HL conditions. The UV-B-dependent expression of NDs genes has been found to be more cooperative with AOX1a at the mRNA level than the expression of UCPs.¹⁶

The expressions of NDB1, NDB3, NDC1, and UCP3 increased in the AS-12 and/or the WT lines but did not change or were down-regulated in the XX-2 line (Figure 1). This suggests that the expressions of these genes are induced in a complementary manner to AOX1a under the HL conditions. The promoters of these genes had higher or lower numbers in CAREs in relation to the AOX1a promoter. The expressions of NDC1, NDB4, and UCP4 were very low in all lines but their promoters contained different numbers of CAREs in common with the AOX1a promoter. In this experiment, the expression of NPhP genes under the different light conditions did not always depend on the number of CAREs in common with AOX1a in their promoters, but clearly depended on the level of AOX1a expression. In addition, the majority of NDs’ genes (five of the seven genes studied) belong to the same large cluster as AOX1a. Enhanced expressions of most genes in terms of NPhPs components in the antisense line suggest that these genes may respond to HL by compensating for the suppression of AOX1a. Altogether, these results indicate that there is an interaction between genes encoding mitochondrial non-phosphorylating proteins both at the level of the transcriptional response and at the level of the formation (convergence) of signaling pathways.¹⁷

A higher basal expression of certain genes (NDA2, NDB1, NDB3, UCP1, and UCP2) in AS-12 plants (0 h) was observed (Figure 1). This indicates that suppression of AOX can induce defenses that ‘overcompensate’ for the lack of AOX. Such ‘overcompensation’ has been observed in other studies ,^16,29,31,32 as well as those involving the manipulation of components of the ROS network ,⁶⁸ suggesting that this may be a common phenomenon. It is thought that AOX suppression enhances a mitochondrial stress-signaling pathway, which leads to an increase in the ROS-scavenging capacity of the cell.

Interestingly, both AOX1a-transformed lines had significantly lower ROS production levels compared with the WT, both in the beginning of the experiment and under light conditions (Figure 4). The response we observed of the XX-2 plants to HL conditions confirms that overexpression of AtAOX1a substantially reduces ROS production and mitigates stress impacts.^16,23,26 In the AS-12 line, higher basal expressions of several NPhPs genes were observed. This can be termed an ‘overcompensation effect’ for AOX suppression in response to higher but not high stress-inducing irradiance conditions. It has been shown that AOX suppression enhances the mitochondrial stress-signaling pathway’s ability to increase the ROS-scavenging capacity of the cell, as well as, possibly, other acclimation responses, following stress.^16,31,32 There is also evidence that the absence of AtAOX1a leads to a decrease in the ability of plants to withstand oxidative stress, especially under severe conditions.^10,25,27,30 The responses of AOX antisense or knockdown lines are obviously determined by signaling pathways depending on the nature, duration, and intensity of the applied stress factor.

Figure 4.

Levels of lipid peroxidation presented as TBARS content (A), superoxide production (B), and hydrogen peroxide concentrations (C) in leaves of wild type (Col-0), XX-2, and AS-12 Arabidopsis thaliana plants grown in control (at 90 μmol m⁻² s⁻¹, i.e. 0 h) and in higher light conditions (at 400 μmol m⁻² s⁻¹, i.e. 2–8 h). Data are presented as mean values ± SE (n = 4–6) taken during three independent experiments. Significant differences between mean values (ANOVA, Duncan’s test, P < 0.05) are indicated by different letters

Clearly, changes in the level of AOX1a expression affected the expression of other AOX genes and genes encoding NPhPs components and, ultimately, determined the level of oxidative reactions. The AOX1a antisense line displayed a clear compensatory effect by induction of NPhPs genes expression showing cooperative and complementarily functioning of the NPhPs components. All NPhPs genes share, in common with AOX1a, light- and stress-related CAREs in their promoters and the majority of NDs’ genes belong to the same large cluster as AOX1a. However, the expression of respiratory genes does not always depend on the level of AOX1a expression. This suggests multiple combinations of signaling pathways involved in NPhPs gene expression. In this way, applying genome engineering techniques to understand the role of certain predicted CAREs in the AtAOX1a promoter in relation to light-dependent regulation of the expressions of AOX1a and genes of other NPhPs components should be the focus of further research.

Funding Statement

This reported study was funded by RFBR (Russian Foundation for Basic Research) according to the research project №19-04-00476 А and partially supported by the grant from The Ministry of Science and Higher Education of the Russian Federation (No. AAAA-A17-117033010038-7).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.