Chihiro K. Watanabe

The lack of alternative oxidase at low temperature leads to a disruption of the balance in carbon and nitrogen metabolism, and to an up-regulation of antioxidant defence systems in Arabidopsis thaliana leaves

Alternative oxidase (AOX) catalyses the ATP-uncoupling cyanide (CN)-resistant pathway. In this study, our aim was to clarify the physiological role of AOX at low temperature. We examined the effect of low-temperature treatment on CN-resistant respiration (CN-resistant R) and on the transcription of respiratory components in wild-type (WT) and aox1a knock-out transgenic (aox1a) Arabidopsis thaliana plants. In WT leaves, the expression of AOX1a mRNA was strongly induced by the low-temperature treatment, and thus CN-resistant R increased during low-temperature treatment. In aox1a, the CN-sensitive respiration, and the expression of NDB2 and UCP1 were increased compared with WT. We compared several physiological parameters between WT and aox1a. Low-temperature treatment did not result in a visible phenotype to distinguish aox1a from WT. In aox1a, several antioxidant defence genes were induced, and the malondialdehyde content was lower than in WT. Starch content and a ratio of carbon to nitrogen were higher in aox1a than in WT. Our results indicate that a lack of AOX was linked to a difference in the carbon and nitrogen balance, and an up-regulation of the transcription of antioxidant defence system at low temperature. It is likely that AOX is a necessary component in antioxidant defence mechanisms and for the control of a balanced metabolism.

Influence of Chloroplastic Photo-Oxidative Stress on Mitochondrial Alternative Oxidase Capacity and Respiratory Properties: A Case Study with Arabidopsis yellow variegated 2

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been demonstrated that leaf AOX is up-regulated under high-light (HL) conditions, the in vivo mechanism of AOX up-regulation by light is still unknown. In the present study, we examined whether the photo-oxidative stress in the chloroplast modulates mitochondrial respiratory properties, especially the AOX capacity, using Arabidopsis leaf-variegated mutant yellow variegated 2 (var2) and exposing plants to HL. var2 mutants lack FtsH2 metalloprotease required for the repair of damaged PSII. Indeed, var2-1 suffered from photo-oxidative stress even before the HL treatments. While the activities of tricarboxylic acid cycle enzymes and cytochrome c oxidase in var2-1 were almost identical to those in the wild type, the amount of AOX protein and the CN-resistant respiration rate were higher in var2-1. Real-time PCR analysis revealed that HL treatment induced the expression of some energy-dissipating respiratory genes, including AOX1a, NDB2 and UCP5, more strongly in var2-1. Western blotting using var2-1 leaf extracts specific to green or white sectors, containing functional or non-functional photosynthetic apparatus, respectively, revealed that more AOX protein was induced in the green sectors by the HL treatment. These results indicate that photo-oxidative stress by excess light is involved in the regulation of respiratory gene expression and the modulation of respiratory properties, especially the AOX up-regulation.

Nitrate Addition Alleviates Ammonium Toxicity Without Lessening Ammonium Accumulation, Organic Acid Depletion and Inorganic Cation Depletion in Arabidopsis thaliana Shoots

When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.

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.

Physiological impact of mitochondrial alternative oxidase on photosynthesis and growth in Arabidopsis thaliana

The mitochondrial alternative oxidase (AOX) has been suggested to have a beneficial role in illuminated leaves, but its function has not yet been fully elucidated. In this study, we investigated the effects of a knockout of the AOX1a gene on photosynthesis and growth under several light conditions in Arabidopsis thaliana. The AOX-deficient aox1a mutant showed a lowered operating efficiency of photosystem II and an enhanced activity of cyclic electron transport around photosystem I (CET-PSI) at high irradiance. To further address the physiological association of AOX with CET-PSI, we crossed aox1a with the pgr5 mutant, which is impaired in CET-PSI activity. In the pgr5 mutant background, AOX deficiency did not affect the apparent photosynthetic efficiency, indicating that the direct contribution of AOX to photosynthesis is not so large compared with CET-PSI. Nevertheless, the growth of the aox1a pgr5 double mutant was significantly impaired depending on the light intensity under growth conditions. The possibility of a synergistic function of AOX with CET-PSI in supporting plant growth is discussed.

Effects of AOX1a Deficiency on Plant Growth, Gene Expression of Respiratory Components and Metabolic Profile Under Low-Nitrogen Stress in Arabidopsis thaliana

Expression of alternative oxidase (AOX) and cyanide (CN)-resistant respiration are often highly enhanced in plants exposed to low-nitrogen (N) stress. Here, we examined the effects of AOX deficiency on plant growth, gene expression of respiratory components and metabolic profiles under low-N stress, using an aox1a knockout transgenic line (aox1a) of Arabidopsis thaliana. We exposed wild-type (WT) and aox1a plants to low-N stress for 7 d and analyzed their shoots and roots. In WT plants, the AOX1a mRNA levels and AOX capacity increased in proportion to low-N stress. Expression of the genes of the components for non-phosphorylating pathways and antioxidant enzymes was enhanced, but differences between WT and aox1a plants were small. Metabolome analyses revealed that AOX deficiency altered the levels of certain metabolites, such as sugars and sugar phosphates, in the shoots under low-N stress. However, the carbon (C)/N ratios and carbohydrate levels in aox1a plants were similar to those in the WT under low-N stress. Our results indicated that the N-limited stress induced AOX expression in A. thaliana plants, but the induced AOX may not play essential roles under stress due to low-N alone, and the C/N balance under low-N stress may be tightly regulated by systems other than AOX.

Effects of elevated CO2 on levels of primary metabolites and transcripts of genes encoding respiratory enzymes and their diurnal patterns in Arabidopsis thaliana: possible relationships with respiratory rates.

Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates.

Ammonium-dependent respiratory increase is dependent on the cytochrome pathway in Arabidopsis thaliana shoots.

Oxygen uptake rates are increased when concentrated ammonium instead of nitrate is used as sole N source. Several explanations for this increased respiration have been suggested, but the underlying mechanisms are still unclear. To investigate possible factors responsible for this respiratory increase, we measured the O₂ uptake rate, activity and transcript level of respiratory components, and concentration of adenylates using Arabidopsis thaliana shoots grown in media containing various N sources. The O₂ uptake rate was correlated with concentrations of ammonium and ATP in shoots, but not related to the ammonium assimilation. The capacity of the ATP-coupling cytochrome pathway (CP) and its related genes were up-regulated when concentrated ammonium was sole N source, whereas the ATP-uncoupling alternative oxidase did not influence the extent of the respiratory increase. Our results suggest that the ammonium-dependent increase of the O₂ uptake rate can be explained by the up-regulation of the CP, which may be related to the ATP consumption by the plasma-membrane H+ -ATPase.

Effects of Elevated Atmospheric CO2 on Primary Metabolite Levels in Arabidopsis thaliana Col-0 Leaves: An Examination of Metabolome Data

Elevated atmospheric CO(2) concentrations ([CO(2)]) affect primary metabolite levels because CO(2) is a direct substrate for photosynthesis. In several studies, the responses of primary metabolite levels have been examined using Arabidopsis thaliana leaves, but these results have not been comprehensively discussed. Here, we examined metabolome data for A. thaliana accession Col-0 leaves that were grown at elevated [CO(2)] with sufficient nitrogen (N) nutrition. At elevated [CO(2)], starch, monosaccharides and several major amino acids accumulated in leaves. The degree of accumulation depended on whether the rooting medium contained NH(4) (+) or only NO(3) (-). Because low N conditions induce an increase in carbohydrates similar to that of elevated [CO(2)], we compared the responses of primary metabolite levels between elevated [CO(2)] and low N conditions. Levels of the tricarboxylic acid (TCA) cycle-associated organic acids and major amino acids decreased with low N, but not with elevated [CO(2)]. Even at elevated [CO(2)], the low N induced the decreases in the levels of organic acids and major amino acids. A small sink size also affects the primary metabolite response patterns in leaves under elevated [CO(2)] conditions. Thus, care is necessary when interpreting primary metabolite changes in leaves of field-grown plants.

Sink–Source Balance and Down-Regulation of Photosynthesis in Raphanus sativus: Effects of Grafting, N and CO2

To clarify whether excessive accumulation of total non-structural carbohydrate (TNC) causes down-regulation of photosynthesis in Raphanus sativus, we manipulated sink-source balance to alter TNC levels in source leaves and examined its effects on photosynthetic characteristics, whole-plant biomass allocation and anatomical characteristics of leaves and petioles. Comet and Leafy varieties with large and small hypocotyls were reciprocally grafted to change hypocotyl sink strength. They were grown at high or low nitrogen (N) availability and at elevated or ambient CO2. Maximum photosynthetic rate, which was highly correlated with Rubisco and leaf N contents, was hardly correlated with TNC across the grafting combinations and growth conditions. Biomass allocation to petioles and hypocotyls and accumulation of TNC in each organ were significantly higher at low N. TNC and structural carbohydrates such as cellulose and hemicellulose were higher and the proportion of intercellular air space in source leaves was lower at low N and elevated CO2. We conclude that excess TNC does not cause severe down-regulation of photosynthesis, and cell walls and petioles are also major carbohydrate sinks responding to changes in sink-source and carbon-nitrogen balances, which contribute to alleviating further accumulation of TNC to avoid its negative effects in source leaves.