Keisuke Yoshida

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.

Differential gene expression profiles of the mitochondrial respiratory components in illuminated Arabidopsis leaves.

Plant mitochondria have multiple energy-dissipating components in the respiratory chain. It is known that these components are induced under several stress conditions. Here we examined whether the gene expression pattern and its regulatory mechanism under high light (HL) conditions are different among the respiratory components in Arabidopsis leaves. Alternative oxidase (AOX) gene expression (AOX1a and AOX1c) and amount of protein were elevated after exposure to HL. In addition to AOX, the expression of other respiratory genes, including NDA1, NDB2, NDC1, UCP1, UCP5, COX6b and CI76, was also induced by HL. NDB2 was co-expressed with AOX1a, but other HL-induced genes showed a distinct expression pattern. Manipulation of photosynthesis or respiration using several chemicals revealed that while the expression of AOX1a and NDB2 was mainly induced by inhibition of the respiratory chain, NDA1 expression was affected by photosynthesis-related reactive oxygen species. The expression of AOX1c, NDC1, COX6b and CI76 was not induced by these manipulations. When plants were exposed to HL under a high CO(2) environment, the expression of several respiratory genes was more strongly induced, suggesting that modulations of cellular carbon status by elevated photosynthesis are involved in respiratory gene expression. Based on these results, we propose a mechanistic model of respiratory gene expression in illuminated leaves.

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.

Simultaneous Determination of In Vivo Plastoquinone and Ubiquinone Redox States by HPLC-Based Analysis

For a better understanding of the metabolic interaction between chloroplasts and mitochondria, it is important to analyze the in vivo redox states of the electron transport chains in both organelles at the same time. For this purpose, we devised an HPLC-based measurement system simultaneously analyzing plastoquinone (PQ) and ubiquinone (UQ) contents and redox states. Using this system, we discovered that, in addition to PQ, the reduction levels of UQ were elevated under high-light conditions. We also provide direct evidence that mitochondrial alternative oxidase contributes to alleviate UQ over-reduction under such conditions.

Damage characterization and numerical modeling of titanium matrix composites subjected to low-velocity impact for landing gear application

Titanium matrix composites (TMCs) is considered as a candidate material for landing gear structures of the aircraft, which requires the damage tolerant consideration. In order to investigate the damage behavior of SiC-fiber/titanium matrix composites subjected to foreign object impact, low-velocity impact experiments are conducted. Finite element modeling to simulate the damage behavior of TMCs is developed considering the plastic deformation and the damages. Numerical results of mechanical response and damage behavior are compared with experimental results. It is shown that the developed modeling can capture the impact damage of TMCs. In the last part of the present study, a prototype of TMC landing gear structure is fabricated. Impact damage behavior and residual compressive strength are evaluated, and the results are compared with numerical simulation.

Functional Analysis of Mitochondrial Respiratory Chain as a Dissipation System of Excess Light Energy

Alternative oxidase (AOX), the unique terminal oxidase in plant mitochondria, catalyzes the energy-wasteful cyanide-resistant respiration. Although it has been suggested that AOX would prevent chloroplast over-reduction via the efficient dissipation of excess reducing equivalents, its direct evidence in the physiological context has been lacking. In the present study, we assessed this possibility using Arabidopsis mutants defective in the cyclic electron flow around PSI. In these mutants, AOX was 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. The presented results indicate that AOX can dissipate the excess light energy and serve in efficient photosynthesis.