Takushi Hachiya

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.

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.

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.

High CO2 Triggers Preferential Root Growth of Arabidopsis thaliana Via Two Distinct Systems Under Low pH and Low N Stresses

Biomass allocation between shoots and roots is an important strategy used by plants to optimize growth in various environments. Root to shoot mass ratios typically increase in response to high CO2, a trend particularly evident under abiotic stress. We investigated this preferential root growth (PRG) in Arabidopsis thaliana plants cultivated under low pH/high CO2 or low nitrogen (N)/high CO2 conditions. Previous studies have suggested that changes in plant hormone, carbon (C) and N status may be related to PRG. We therefore examined the mechanisms underlying PRG by genetically modifying cytokinin (CK) levels, C and N status, and sugar signaling, performing sugar application experiments and determining primary metabolites, plant hormones and expression of related genes. Both low pH/high CO2 and low N/high CO2 stresses induced increases in lateral root (LR) number and led to high C/N ratios; however, under low pH/high CO2 conditions, large quantities of C were accumulated, whereas under low N/high CO2 conditions, N was severely depleted. Analyses of a CK-deficient mutant and a starchless mutant, in conjunction with sugar application experiments, revealed that these stresses induce PRG via different mechanisms. Metabolite and hormone profile analysis indicated that under low pH/high CO2 conditions, excess C accumulation may enhance LR number through the dual actions of increased auxin and decreased CKs.

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.

Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants

Nitrogen (N) availability is a major factor determining plant growth and productivity. Plants acquire inorganic N from the soil, mainly in the form of nitrate and ammonium. To date, researchers have focused on these N sources, and demonstrated that plants exhibit elaborate responses at both physiological and morphological levels. Mixtures of nitrate and ammonium are beneficial in terms of plant growth, as compared to nitrate or ammonium alone, and therefore synergistic responses to both N sources are predicted at different steps ranging from acquisition to assimilation. In this review, we summarize interactions between nitrate and ammonium with respect to uptake, allocation, assimilation, and signaling. Given that cultivated land often contains both nitrate and ammonium, a better understanding of the synergism between these N sources should help to identify targets with the potential to improve crop productivity.

Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

Integrative response of plant mitochondrial electron transport chain to nitrogen source

Nitrogen (N) availability is widely known as a determinant of plant growth and respiration rate. However, less attention has been paid to the effect of the type of N source (nitrate, nitrite or ammonium) on the respiratory system. This review summarizes the latest findings on this topic, with an emphasis on the effect of ammonium and nitric oxide (NO) on the respiratory system, and the physiological role of alternative oxidase (AOX). First, concentrated ammonium has been found to increase plant respiration rate (ammonium-dependent respiratory increase, ARI). We will introduce two hypotheses to explain ARI, futile ammonium cycling and excess reducing equivalents, and verify the validity of each hypothesis. We suggest that these two hypotheses are not necessarily mutually exclusive. Second, gene expression of AOX is suppressed when N is predominately available as nitrate instead of ammonium. We will discuss possible signaling pathways leading to this expression pattern. Third, while AOX expression is induced by NO, AOX activity itself is insensitive to NO. In contrast, activity of cytochrome c oxidase (COX) is sensitive to NO. We outline the NO production pathway, focusing on nitrite-dependent NO production, and discuss the physiological significance of the fact that AOX activity is insensitive to NO. Finally, this review aims to build an integrated scheme of the respiratory response to the type of N source, considering leaves in high light conditions or hypoxic roots.

Effect of growth temperature and total non-structural carbohydrate accumulation on growth coefficient in Petunia x hybrida petals.

It is widely believed that growth coefficient [g, respiratory cost consumed for synthesis of a unit dry weight (DW)] is independent of growth temperature. However, some studies using regression analysis indicated an increase in g at high temperatures. These two conflicting conclusions may be affected by the differences in total non-structural carbohydrate (TNC) accumulation between growth temperatures. Here, we evaluated whether g is independent of growth temperature in Petunia x hybrida petal after excluding TNC from DW. We also examined changes in g during petal development. We measured amounts of carbon, nitrogen, minerals and TNC and theoretically estimated construction cost and g. The results showed that the estimated g greatly changed with age and differed between growth temperatures. The estimated g was markedly increased by excluding TNC from DW. The effect of accumulation of TNC on the estimated g was observed at later stage of petal development. At earlier stage, the estimated g depended on the N amount. These results indicate that g changes with growth temperature and suggest that TNC accumulation must be taken into account in the estimation.