Junichi Mano

Thioredoxin Deficiency Causes the Constitutive Activation of Yap1, an AP-1-like Transcription Factor in Saccharomyces cerevisiae

Yap1 is a transcription factor that responds to oxidative stress in Saccharomyces cerevisiae. The activity of Yap1 is regulated at the level of its intracellular localization, and a cysteine-rich domain at the C terminus of Yap1 is involved in this regulation. We investigated the effects of redox-regulatory proteins, thioredoxin and glutaredoxin, on the regulation of Yap1, using the deficient mutants of these thiol-disulfide oxidoreductases. In the thioredoxin-deficient mutant (trx1Δ/trx2Δ), Yap1 was constitutively concentrated in the nucleus and the level of expression of the Yap1 target genes was high under normal conditions, while this was not the case for the glutaredoxin-deficient mutant (grx1Δ/grx2Δ). No distinct difference was observed in the levels of Yap1 protein between the wild type andtrx1Δ/trx2Δ. The constitutive activation of Yap1 in trxΔ/trx2Δ was observed under aerobic conditions but not under anaerobic conditions. These findings suggest that thioredoxin has negative effects on this regulation via the redox states. We also show the synthetic lethality betweenyap1Δ and trx1Δ/trx2Δ mutation, but theyap1Δ/grx1Δ/grx2Δ triple mutant was viable, suggesting a difference of the functions between thioredoxin and glutaredoxin and a genetic interaction between Yap1 and thioredoxin in vivo.

Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR

Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O2- in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP(+) reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2.

Protection against Photooxidative Injury of Tobacco Leaves by 2-Alkenal Reductase. Detoxication of Lipid Peroxide-Derived Reactive Carbonyls

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the α,β-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the α,β-unsaturated bond of the photoproduced reactive carbonyls.

The Involvement of Lipid Peroxide-Derived Aldehydes in Aluminum Toxicity of Tobacco Roots

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic α,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl3 on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl3 treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl3 treatment, the roots of AER-OE plants accumulated Al and H2O2 to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl3 treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.

Reactive carbonyl species: Their production from lipid peroxides, action in environmental stress, and the detoxification mechanism

Accumulation of lipid peroxide-derived aldehydes and ketones is a ubiquitous event in oxidative stress. The toxicity of these carbonyls, especially the α,β-unsaturated carbonyls (reactive carbonyls; RCS), in environmental-stressed plants has been demonstrated by several independent research groups, on the basis of the results that overexpression of different carbonyl-detoxifying enzymes commonly improved tolerance of the transgenic plants against environmental stresses. A positive correlation between the level of carbonyls and the stress-induced damage in these plants proves the cause-effect relationship between carbonyls and the cell injury. Comprehensive analysis of carbonyls has revealed that dozens of distinct RCS including highly toxic acrolein and 4-hydroxy-2-nonenal are contained at nmol/g fresh weight levels in the tissues of non-stressed plants. Stress treatments of plants increase the levels of these RCS, likely reaching a sub-mM order, but in the transgenic plants overproducing RCS-detoxifying enzymes, their increase is significantly suppressed. Immunological analyses have demonstrated that in non-stressed cells several proteins are modified by RCS and the extent of modification is increased on stresses. In heat-stressed leaves, the inactivation of the oxygen-evolving complex was associated with selective modification of OEC33 protein and photosystem II core proteins. RCS consume glutathione and inactivate various enzymes in chloroplasts and mitochondria, thereby accelerating oxidative stress status. Thus RCS, formed downstream of reactive oxygen species (ROS), act in a way biochemically distinct from that of ROS and play critical roles in the plant responses to oxidative stress.

Differential Metabolisms of Green Leaf Volatiles in Injured and Intact Parts of a Wounded Leaf Meet Distinct Ecophysiological Requirements

Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. 13C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,β-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf.

Ascorbate in thylakoid lumen as an endogenous electron donor to Photosystem II: Protection of thylakoids from photoinhibition and regeneration of ascorbate in stroma by dehydroascorbate reductase

Photoinhibition of the electron transport activity from tyrosine Z (YZ) in PS II to NADP+in Tris-treated thylakoids was suppressed by electron donation with either diphenylcarbazide or ascorbate (AsA) during the photoinhibition treatment. This suggests that AsA prevents donor side-induced photoinhibition in vivo as an endogenous donor. AsA in the lumen is photooxidized to monodehydroascorbate (MDA) in Tris-treated thylakoids, as detected by electron spin resonance spectrometry, but not in oxygenic thylakoids. Redox analysis of pyridine nucleotide in the presence of either MDA reductase or dehydroascorbate (DHA) reductase showed that the MDA photoproduced in the lumen is disproportionated to AsA and DHA, and the DHA leaking into the stroma is reduced to AsA by DHA reductase. No leakage of MDA through the thylakoid membrane was observed. Thus, the DHA-reducing enzyme system is indispensable in maintaining AsA concentrations in chloroplasts.

Differential electron flow around photosystem I by two C4-photosynthetic-cell-specific ferredoxins

In the C4 plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle‐sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2‐9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 μmol/m2/s), whereas KM2‐9 grew at a normal rate but exhibited a nitrogen‐deficient phenotype. Measurement of photosynthetic O2 evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2‐9. After starvation of the cells in darkness, the P700 oxidation level under far‐red illumination increased significantly in TM202. However, it remained low in KM2‐9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP+ decreased in KM2‐9 as compared with TM202. These results demonstrated that the two cell type‐specific ferredoxins differentially modulate electron flow around photosystem I.

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.

A novel l-isoleucine metabolism in Bacillus thuringiensis generating (2S,3R,4S)-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid

Abstract4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for l-isoleucine (l-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2S,3R,4S)-HIL. The (2S,3R,4S)-HIL was oxidized to (2S,3R)-AMKP by a NAD+-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B12 antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP.