Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in Arabidopsis

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide‐resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide‐resistant respiration was also accompanied by the increase of plastid NADPH/NADP⁺ ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin–Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX‐derived plastidial NADPH/NADP⁺ ratio change. Further, our results thus revealed a molecular mechanism of chloroplast–mitochondria interactions.     

Distinct roles of alternative oxidase pathway during the greening process of etiolated algae

The vital function of mitochondrial alternative oxidase(AOX) pathway in optimizing photosynthesis during plant de-etiolation has been well recognized. However, whether and how AOX impacts the chloroplast biogenesis in algal cells remains unclear. In the present study, the role of AOX in regulating the reassembly of chloroplast in algal cells was investigated by treating Auxenochlorella protothecoides with salicylhydroxamic acid(SHAM), the specific inhibitor to AOX, in the heterotrophy to autotrophy transition process. Several lines of evidences including delayed chlorophyll accumulation, lagged reorganization of chloroplast structure, altered PSI/PSII stoichiometry, and declined photosynthetic activities in SHAM treated cells indicated that the impairment in AOX activity dramatically hindered the development of functioning chloroplast in algal cells. Besides, the cellular ROS levels and activities of antioxidant enzymes were increased by SHAM treatment, and the perturbation on the balance of NAD~+/NADH and NADP~+/NADPH ratios was also observed in A. protothecoides lacking AOX activity, indicating that AOX was essential in promoting ROS scavenging and keeping the redox homeostasis for algal chloroplast development during greening. Overall, our study revealed the essentiality of mitochondrial AOX pathway in sustaining algal photosynthetic performance and provided novel insights into the physiological roles of AOX on the biogenesis of photosynthetic organelle in algae.     

Up-regulation of mitochondrial alternative oxidase concomitant with chloroplast over-reduction by excess light

Alternative oxidase (AOX), the unique terminal oxidase in plant mitochondria, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been suggested that AOX might prevent chloroplast over-reduction through the efficient dissipation of excess reducing equivalents, direct evidence for this in the physiological context has been lacking. In this study, we examined the mitochondrial respiratory properties, especially AOX, connected to the accumulation of reducing equivalents in the chloroplasts and the activities of enzymes needed to transport the reducing equivalents. We used Arabidopsis thaliana mutants defective in cyclic electron flow around PSI, in which the reducing equivalents accumulate in the chloroplast stroma due to an unbalanced ATP/NADPH production ratio. These mutants showed higher activities of the enzymes needed to transport the reducing equivalents even in low-light growth conditions. The amounts of AOX protein and CN-resistant respiration in the mutants were also higher than those in the wild type. After high-light treatment, AOX, even in the wild type, was preferentially up-regulated concomitant with the accumulation of reducing equivalents in the chloroplasts and an increase in the activities of enzymes needed to transport reducing equivalents. These results indicate that AOX can dissipate the excess reducing equivalents, which are transported from the chloroplasts, and serve in efficient photosynthesis.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

烟草叶片中呼吸电子传递途径在缓解叶绿体PSⅡ光抑制中的作用

前期研究发现线粒体交替氧化酶(AOX)呼吸途径对叶绿体光系统II(PSII)的光抑制有明显的缓解作用。线粒体内另一条呼吸途径——细胞色素氧化酶(COX)呼吸途径是否也具有光保护作用尚不清楚。该文通过荧光快速诱导动力学和荧光淬灭分析,解析了烟草(Nicotiana tabacum)叶片中COX途径对PSII光保护的贡献及其与AOX途径的关系。结果表明,强光处理后PSII活性在所有叶片中均下降。AOX途径受抑明显加速了叶片PSII活性的下降。而当COX途径受抑后,叶片PSII活性的下降与水处理的对照叶片无明显差异。当AOX途径与COX途径同时受抑时,叶片PSII活性的下降比单独抑制AOX途径时更严重。此外,呼吸电子传递受抑均导致叶片非光化学淬灭(NPQ)增加,AOX途径受抑导致的NPQ上调比COX途径受抑时更明显,AOX和COX途径同时受抑时NPQ的增幅最大。上述结果表明,烟草叶片中COX途径和AOX途径均参与PSⅡ的光保护。当COX途径受抑时,其光保护功能可以被AOX途径和NPQ补偿,而AOX途径的光保护作用不能被COX途径和NPQ完全补偿。     

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities)

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.     

Involvement of photobleaching and inhibition of protochlorophyll(ide) accumulation in tentoxin effects on greening of mung bean seedlings

Several types of evidence indicate that tentoxin-caused reduction of chlorophyll accumulation in greening primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] is due to both photobleaching and decreased protochlorophyll(ide) synthesis. Greening was greater under dim (2.5 μmol m_-2 s_-1) far-red or white light than under bright (180 to 200 μmol m_-2 s_-1) white light in tentoxin-treated tissues, whereas there was a positive correlation between fluence rate and greening in control tissues. Under continuous white light (100 μmol m_-2 s_-1) chorophyll(ide) accumulation was slower in tentoxin-treated than in control tissues. This was caused by greater photobleaching of newly formed chlorophyll(ide), as well as by decreased protochlorophyll(ide) synthesis. Photobleaching did not affect protochlorophyll(ide) synthesis in control or tentoxin-treated tissues. Chlorophyll(ide) was less stable in tentoxin-treated than in control tissues during a 24 h period of darkness. Plastids of tentoxin-treated tissues had all of the chlorophyll-proteins of control plants. Etioplasts of tentoxin-treated plants contained normal galactolipid contents, but galactolipids in these plants were greatly reduced in white light. Reduced chlorophyll accumulation caused by tentoxin is apparently the result of both photodestruction and of reduced synthesis of chlorophyll.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

Increased respiratory restriction during phosphate-limited growth in transgenic tobacco cells lacking alternative oxidase

We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

Phytochrome Control of Chlorophyll Content in Mature Attached Leaves of Petunia axillaris

Petunia axillaris plants were grown under white light (photoperiod10 h). Pulses of end-of-day far-red light, but not red lightat the end of the day slowed down the accumulation of chlorophyllin expanding, greening leaves and reduced chlorophyll contentin mature leaves. The chlorophyll a / b ratio was unaffected.Low phytochrome photoequilibria reduced chlorophyll contentin mature leaves without affecting leaf area or d. wt. Thiseffect occurred even before senescence was triggered in thecontrol plants having high phytochrome photoequilibria     

Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress

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.     

Cellular functions of cardiolipin in yeast

Cardiolipin (CL), the signature lipid of mitochondria, plays a critical role in mitochondrial function and biogenesis. The availability of yeast mutants blocked in CL synthesis has facilitated studies of the biological role of this lipid. Perturbation of CL synthesis leads to growth defects not only during respiratory growth but also under conditions in which respiration is not essential. CL was shown to play a role in mitochondrial protein import, cell wall biogenesis, aging and apoptosis, ceramide synthesis, and translation of electron transport chain components. The genetic disorder Barth syndrome (BTHS) is caused by mutations in the tafazzin gene resulting in decreased total CL levels, accumulation of monolysocardiolipin (MLCL), and decreased unsaturated fatty acyl species of CL. The variation in clinical presentation of BTHS indicates that other physiological factors play a significant role in modifying the phenotype resulting from tafazzin deficiency. Elucidating the functions of CL is expected to shed light on the role of this important lipid in BTHS and other disorders of mitochondrial dysfunction.     

The effect of simulated microgravity on formation of the pigment apparatus in etiolated barley seedlings

The effect of horizontal clinorotation on the dynamics of the accumulation of the main photosynthetic pigments in the greening of 6-day-old etiolated barley seedlings has been studied. The content of protochlorophillide, the direct precursor of chlorophyll a, in clinorotated seedlings in the dark was 9–20% lower than in the control group. After exposure of barley seedlings to light for 12 h under clinorotation, chlorophyll accumulation lagged behind the control by 45% and reached the control value after 48–72 h. The total content of carotenoids increased many fold during greening; at the first stage the carotenoid level in clinorotated seedlings was less than in the control. The synthesis rates of δ-aminolevulinic acid and δ-aminolevulinate dehydratase activity in clinorotated seedlings were slower than in the control after 24 h of greening and after 72 h of greening reaching the control values. The activity of Mg-protoporphyrin IX chelatase catalyzing the incorporation of Mg ions in the structure of chlorophyll a, did not change when exposed to clinorotation. The results we obtained show inhibition of the initial stages of chlorophyll biosynthesis in the conditions of simulated microgravity. The light, to a certain extent, decreases the negative effect of microgravity on the formation of the photosynthetic apparatus in plants.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of nutrient uptake

When wild type (wt) tobacco ( Nicotiana tabacum L. cv. Petit Havana SR1) suspension cells were grown under phosphate (P) limitation, they contained large amounts of mitochondrial alternative oxidase (AOX). When these cells were resupplied with P, there was a large, immediate and sustained stimulation of respiration to support a period of rapid P uptake. Two lines of evidence suggest that the abundant level of AOX present in wt cells contributed to this stimulated rate of respiration. First, when P-limited transgenic antisense tobacco cells (AS8) lacking AOX were resupplied with P, the stimulation of respiration was much less dramatic even though these cells displayed similar rates of P uptake. Second, while the stimulated rate of respiration in AS8 cells was insensitive (as expected) to the AOX inhibitor n -propyl gallate (nPG), much of the stimulated rate of respiration in wt cells could be inhibited by nPG. Given the non-phosphorylating nature of AOX respiration, wt cells required higher rates of electron transport to O₂ than AS8 cells to support similar rates of P uptake. The utilization of AOX by wt cells during P uptake was apparently not occurring because the cytochrome (Cyt) pathway alone could not fully support the rate of P uptake, as the respiration of cells lacking AOX (either untreated AS8 cells or wt cells treated with nPG) supported similar rates of P uptake as wt cells with abundant AOX. Rather, we provide in vivo evidence that the utilization of AOX during the period of high respiration supporting P uptake was to dampen the mitochondrial generation of active oxygen species (AOS).