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The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing th...
Citations
... Mitochondrial alternative oxidase 1a whose own mTP was replaced with the cTP of rubisco small subunit (RbcS) was successfully localized to chloroplasts and restored a plastid terminal oxidase activity in the immutans mutants of Arabidopsis. 13 A transformation of the cTP-chloroplast ribosomal protein S12 (rps12) fusion into the nuclear genome rescued the Arabidopsis ppr4 mutant that is impaired in the correct splicing of chloroplast rps12 intron 1b. 14 Similarly, mitochondrial NADH dehydrogenase subunit 7 (NAD7) fused with the mTP of Arabidopsis F1-ATPase γ-subunit (pFAγ) restored the phenotype of slow growth 3 mutant that is defective in the splicing of mitochondrial nad7 intron 2. 15 Moreover, artificial chloroplast targeting of phytochelatin synthase enhanced the tolerance of Arabidopsis against heavy metal stress. ...
The chloroplasts in terrestrial plants play a functional role as a major sensor for perceiving physiological changes under normal and stressful conditions. Despite the fact that the plant chloroplast genome encodes around 120 genes, which are mainly essential for photosynthesis and chloroplast biogenesis, the functional roles of the genes remain to be determined in plant’s response to environmental stresses. Photosynthetic electron transfer D (PETD) is a key component of the chloroplast cytochrome b6f complex. Chloroplast ndhA (NADH dehydrogenase A) and ndhB (NADH dehydrogenase B) interact with photosystem I (PSI), forming NDH-PSI supercomplex. Notably, artificial targeting of chloroplasts-encoded proteins, PETD, NDHA, or NDHB, was successfully relocated from cytosols into chloroplasts. The result suggests that artificial targeting of proteins to chloroplasts is potentially open to the possibility of chloroplast biotechnology in engineering of plant tolerance against biotic and abiotic stresses.
... Природу зв'язку між органелами в клітині та механізми взаємодії дедалі більше досліджують з використанням мутантних особин [50,51]. Для вирішення фундаментальних питань фотосинтезу і вивчення біогенезу хлоропластів останнім часом зростає інтерес до застосування мутантів строкатості [51,52]. Вони складаються із зелених, білих і жовтих секторів, що виникають внаслідок мутації в генах [53]. ...
... High light intensity prevents chloroplast formation and thus reduces Chl concentrations in plants. In contrast, the low light intensity reduces chloroplast numbers per unit area while it increases the size of each chloroplast, ultimately leading to an increase in the Chl concentration (Fu et al., 2012). Moreover, the low light intensity can reportedly increase Chl concentrations in plants (Hou et al., 2010). ...
... However, the increased PTOX did not optimize PSI and PSII in N. tabacum AOX knockdowns after AntA treatment (Alber & Vanlerberghe 2021). Interestingly, AOX1A, AOX2 and other forms of AOX can rescue defects of the immutans (im) variegation mutant of A. thaliana by replacing PTOX activity in the desaturation steps of carotenogenesis (Fu et al. 2012;Wang et al. 2021). It was also found that AOX2 is imported into chloroplasts using its own transpeptide (Fu et al. 2012). ...
... Interestingly, AOX1A, AOX2 and other forms of AOX can rescue defects of the immutans (im) variegation mutant of A. thaliana by replacing PTOX activity in the desaturation steps of carotenogenesis (Fu et al. 2012;Wang et al. 2021). It was also found that AOX2 is imported into chloroplasts using its own transpeptide (Fu et al. 2012). The ability of AOX1A and AOX2 to act as substitutes for PTOX in the physiological and developmental contexts illustrates the plasticity of the photosynthetic apparatus and, as a whole, the photosynthesizing cell. ...
Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX-genes knockout is aimed at revealing the functions of AOX genes. Under unfavorable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists in the absence of AOX1a leading to an increase in defense response mechanisms concomitant with a decrease in reactive oxygen species contents has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of reactive oxygen species and mitochondrial membrane potential in triggering the signal transmitting from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell in response to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of the reduced ascorbate at stable reactive-oxygen-species levels as a result of compensation in AOX1a-suppressed plants is proposed to be a sign of stress development. Obtaining direct evidence on the mechanisms and signaling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signaling pathways.
... All five AOXs can use PQH 2 as a substrate, and AOX1a, AOX1b, and AOX2 may be targeted to chloroplasts [106]. Based on findings obtained and literature data [144,145], Wang et al. [106] hypothesized that AOX1a, AOX1b, and AOX2 might be dually targeted to mitochondria and chloroplasts, while AOX1c and AOX1d are specifically localized in mitochondria. It should be noted, however, that all five Arabidopsis AOXs were predicted to be localized in mitochondria, according to the analysis of targeting peptides [146,147]. ...
Chlororespiration is the uptake of oxygen into the respiratory electron transport chain (ETC) localized in the thylakoid membranes of chloroplasts. The chlororespiratory ETC interacts with photosynthetic electron transport and participates in the non-photochemical reduction/oxidation of the plastoquinone pool (PQP) accompanied by O 2 consumption. The two key thylakoid enzymes in chlororespiration are the plastid-encoded NAD(P)H dehydrogenase complex (NDH) and the nucleus-encoded terminal plastoquinol oxidase (PTOX). The contribution of chlororespiratory electron flux to the total electron flow in non-stressed plants is considered insignificant. In contrast, under abiotic stresses, chlororespiration appears to be triggered, at least in some photosynthetic organisms, acting as a protective alternative electron transport pathway. There is evidence of NDH complex and PTOX increasing their activity and/or abundance when plants experience high light, drought, heat, or low-temperature stresses. Alternative electron transfer to oxygen via PTOX protects PQP from over-reduction under stress conditions. For instance, it was shown that PTOX-dependent electron drainage accounted for up to 30% of total PSII electron flow in salt-stressed plants. PTOX is not bound to the thylakoid membrane in dark-adapted leaves but is associated with it at intense illumination and high transmembrane proton gradient (ΔpH) or membrane potential (Δψ). It was also shown that PTOX is capable of lateral translocation from stromal lamellae to granal thylakoid stacks under salt stress. Such changes in PTOX localization increase the accessibility of the substrate (plastoquinol) and the turnover rate of the enzyme. The available data allow considering PTOX as a possible target for manipulation to increase stress tolerance in sensitive plants.
... Multiple sequence alignment of plastid terminal oxidase (PTOX) revealed a unique CTD domain. (a) Schematic representation of the Arabidopsis thaliana PTOX (AtPTOX) protein, including the transit peptide domain (TP) and the catalytic diiron carboxylate domain (DOX) residues (asterisks), represented by amino acid residues E136, E175, E178, E227, E296 and H299 (based on(Fu et al., 2012). The conserved cysteines (C1, C2 and C3) are present in the DOX domain, whereas C4, C5 and C6 represent the C-terminal domain (CTD) of AtPTOX. ...
Disulfide‐based regulation links the activity of numerous chloroplast proteins with photosynthesis‐derived redox signals. The Plastid Terminal Oxidase (PTOX) is a thylakoid‐bound plastoquinol oxidase that has been implicated in multiple roles in the light and in the dark, which could require different levels of PTOX activity. Here we show that Arabidopsis PTOX contains a conserved C‐terminus domain (CTD) with cysteines that evolved progressively following plants colonization of the land. Furthermore, the CTD contains a regulatory disulfide that is in the oxidized state in the dark and is rapidly reduced, within 5 min, in low, 1‐5 µE*m‐2*s‐1, light intensity. The reduced PTOX form in the light was reoxidized within 15 min after the transition to the dark. Mutation of the cysteines in the CTD prevented the formation of the oxidized form. This resulted in higher levels of reduced plastoquinone when measured at transition to the onset of low light. This is consistent with the reduced state of PTOX exhibiting diminished PTOX oxidase activity under conditions of limiting PQH2 substrate. Our findings suggest that AtPTOX‐CTD evolved to add light‐dependent regulation of PTOX activity for the adaptation of plants to terrestrial conditions.
... AOX is a UQH 2 oxidase in mitochondria, while PTOX is a PQH 2 oxidase in chloroplasts. In vitro enzyme assays showed that PTOX specifically uses PQH 2 as substrate and AOX exclusively uses UQH 2 (Josse et al., 2003;Fu et al., 2012;Yu et al., 2014). ...
... PTOX is also critical for carotenoid biosynthesis since inactivation of PTOX results in a deficiency of phytoene desaturation, a key step in carotenoid biosynthesis (Okegawa et al., 2010;McDonald et al., 2011). Consequently, the Arabidopsis PTOX null mutant, immutans (im), shows a striking light-dependent variegation phenotype due to the absence of protective carotenoids (Wu et al., 1999;Fu et al., 2005Fu et al., , 2009Fu et al., , 2012. In addition, PTOX participates in chloroplast development and plant photomorphogenesis (Foudree et al., 2012;Tamiru et al., 2013). ...
... Besides providing a mitochondrial means to indirectly optimize the chloroplasts energy status, AOX could directly enter chloroplasts and regulate PETC in the thylakoid membrane (Fu et al., 2012). Overexpressing AOX2 in Arabidopsis im rescued the variegation phenotype of the mutant, and AOX2 was found dually targeted to mitochondria and chloroplasts. ...
Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.
... Oxidase AOX (McDonald et al., 2010) and their complementary functions have recently been investigated in plants, where the phenotype of Arabidopsis mutant strains lacking PTOX was recovered when AOX protein was targeted to the plastid (Fu et al., 2012). Besides its role in carotenoid biosynthesis (PTOX is an electron acceptor to phytoene desaturase (Gemmecker et al., 2015), this enzyme is supposed to act as a safety valve for excess of photosynthetic electrons in high light condition, when a high ΔpH would slow down electron transfer at the level of cytochrome b6f activity potentially leading to photodamage of PSII. ...
To produce the energy needed for cell metabolism, eukaryotic photosynthetic organisms rely on two organelles: the chloroplast and the mitochondrion. The former converting light energy into chemical energy, the latter performing cell respiration. Since both organelles have overlapping function, their activities need to be regulated. While in plants and green algae they seem to work overall independently according to environmental conditions, like light and sugar availability, in Diatoms the direct exchange of ATP and NADPH between these two organelles are essential for the cell’s survival. Although the physiology of this energetic crosstalk is well established, the molecular actors of this process are still unknown. During this PhD project, I have selected four candidate proteins, which turned out to play a role the organelles’ cross talk mechanisms. These are transporters predicted to be located within the chloroplast envelope and the inner membrane of the mitochondrion. To understand their physiological role, we compared the photosynthetic performances of the wildtype and the mutant strains with spectroscopic and fluorescence approaches, while the respiration was quantified measuring the oxygen evolution rates.The observed differences suggest that the selected transporters play a role the chloroplast-mitochondrion crosstalk and that other proteins might be involved in this regulative process.The further characterization of these transporters might also validate them as possible targets to improve algal biomass productivity for biotech, promoting the simultaneous use of respiration and photosynthesis (mixotrophy).
... Moreover, an active site, "E(x)6Y", detected by Nakamura was also found in the conserved domain in both subfamilies [37], as were two di-iron coordination motifs named "ExxH", although they were quite different in gene structure and location [9]. This structural consistency might lead to a strong similarity in function in many cases, such as the functional redundancy of the two subfamily proteins of A. thaliana detected in chloroplasts [47]. In contrast to the respiratory ETC in mitochondria, the chlororespiration ETC ...
... Moreover, an active site, "E(x)6Y", detected by Nakamura was also found in the conserved domain in both subfamilies [37], as were two di-iron coordination motifs named "ExxH", although they were quite different in gene structure and location [9]. This structural consistency might lead to a strong similarity in function in many cases, such as the functional redundancy of the two subfamily proteins of A. thaliana detected in chloroplasts [47]. In contrast to the respiratory ETC in mitochondria, the chlororespiration ETC carries a distant terminal oxidase, which is able to transfer electrons from NADH/NADPH to plastoquinone (PQ) [48]. ...
Flower colour and colour patterns are crucial traits for ornamental species; thus, a comprehensive understanding of their genetic basis is extremely significant for plant breeders. The tulip tree (Liriodendron tulipifera Linn.) is well known for its flowers, odd leave shape and tree form. However, the genetic basis of its colour inheritance remains unknown. In this study, a putative plastid terminal oxidase gene (LtuPTOX) was identified from L. tulipifera based on multiple databases of differentially expressed genes at various developmental stages. Then, the full-length cDNA of LtuPTOX was derived from tepals and leaves using RACE (rapid amplification of cDNA ends) approaches. Furthermore, gene structure and phylogenetic analyses of PTOX as well as AOXs (alternative oxidases), another highly similar homologue in the AOX family, were used to distinguish between the two subfamilies of genes. In addition, transient transformation and qPCR methods were used to determine the subcellular localization and tissue expression pattern of the LtuPTOX gene. Moreover, the expression of LtuPTOX as well as pigment contents was investigated to illustrate the function of this gene during the formation of orange bands on petals. The results showed that the LtuPTOX gene encodes a 358-aa protein that contains a complete AOX domain (PF01786). Accordingly, the LiriodendronPTOX and AOX genes were identified as only paralogs since they were rather similar in sequence. LtuPTOX showed chloroplast localization and was expressed in coloured organs such as petals and leaves. Additionally, an increasing pattern of LtuPTOX transcripts leads to carotenoid accumulation on the orange-band during flower bud development. Taken together, our results suggest that LtuPTOX is involved in petal carotenoid metabolism and orange band formation in L. tulipifera. The identification of this potentially involved gene will lay a foundation for further uncovering the genetic basis of flower colour in L. tulipifera.
... The protein samples were separated on a 12% SDS-PAGE gel. Immunoblot analysis against PTOX was done as described previously using a rabbit polyclonal antibody (Fu et al. 2012). Chemiluminescent signals were generated using the ECL Plus reagent (Tanon, China) and visualized using the Tanon-5200 Chemiluminescent Imaging System (Tanon, China). ...
To develop an easy and robust method for creating genetically stable and easily detectable Arabidopsis mutants, we adopted the polycistronic tRNA–gRNA CRISPR/Cas9 (PTG/Cas9) system, a multiplex gene-editing tool in rice, with PTOX as the reporter gene. The PTG/Cas9 system has a great potential in generating large deletions detectable by PCR, which greatly simplifies the laborious work of mutant screening. We constructed a PTOX–PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis. At T1 generation, 24.4% of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus, but no homozygous mutant was found, indicating that gene editing occurred predominantly in somatic cells. After a self-cross propagation, 60% of T1 chimeric plants were able to produce homozygous, heterozygous, or bi-allelic ptox offsprings. Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation. We further targeted five other genes using the same procedure and achieved homozygous Cas9-free mutants with large deletions for all genes within three generations. We established a standard and reliable protocol to generate stable inherited deletion mutants in 2–3 generations along with simple PCR screening methods. We conclude that the rice PTG/Cas9 system is an efficient, easy, and rapid tool to edit genes in Arabidopsis. We propose that it could be applied to other genes in Arabidopsis, and it might have the potential to edit genes in other plant species as well.
