Masahiro Tamoi

Overexpression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth.

Transgenic tobacco plants expressing a cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase targeted to chloroplasts show enhanced photosynthetic efficiency and growth characteristics under atmospheric conditions (360 p.p.m. CO2). Compared with wild-type tobacco, final dry matter and photosynthetic CO2 fixation of the transgenic plants were 1.5-fold and 1.24-fold higher, respectively. Transgenic tobacco also showed a 1.2-fold increase in initial activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) compared with wild-type plants. Levels of intermediates in the Calvin cycle and the accumulation of carbohydrates were also higher than those in wild-type plants. This is the first report in which expression of a single plastid-targeted enzyme has been shown to improve carbon fixation and growth in transgenic plants.

H2O2-triggered Retrograde Signaling from Chloroplasts to Nucleus Plays Specific Role in Response to Stress

Background: Detailed insight into the role of chloroplastic H2O2 in cell signaling is necessary. Results: A large change in gene expression occurred in response to chloroplastic H2O2, resulting in positive and negative effects on the response to stresses. Conclusion: Chloroplastic H2O2 regulates abiotic and biotic stress response. Significance: We provided a new insight into the role of chloroplastic H2O2 in stress response. Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H2O2 levels. Here, to clarify the signaling function of H2O2 derived from the chloroplast, we created a conditional system for producing H2O2 in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H2O2 in HL response. These findings provide a new insight into the role of H2O2-triggered retrograde signaling from chloroplasts in the response to stress in planta.

Arabidopsis chloroplastic ascorbate peroxidase isoenzymes play a dual role in photoprotection and gene regulation under photooxidative stress.

Though two types of chloroplastic ascorbate peroxidase (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H(2)O(2), their physiological significance in the response to photooxidative stress is still under discussion. Here we characterized single mutants lacking either tAPX (KO-tAPX) or sAPX (KO-sAPX). Under exposure to high light or treatment with methylviologen under light, H(2)O(2) and oxidized proteins accumulated to higher levels in both mutant plants than in the wild-type plants. On the other hand, the absence of sAPX and tAPX drastically suppressed the expression of H(2)O(2)-responsive genes under photooxidative stress. Interestingly, the most marked effect of photooxidative stress on the accumulation of H(2)O(2) and oxidized protein and gene expression was observed in the KO-tAPX plants rather than the KO-sAPX plants. The present findings suggest that both chloroplastic APXs, but particularly tAPX, are important for photoprotection and gene regulation under photooxidative stress in Arabidopsis leaves.

Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn2+, Cd2+, and l-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.

Acquisition of a new type of fructose-1,6-bisphosphatase with resistance to hydrogen peroxide in cyanobacteria: molecular characterization of the enzyme from Synechocystis PCC 6803

We have previously described that Synechococcus PCC 7942 cells contain two fructose-1,6-bisphosphatase isozymes, designated F-I and F-II the former belongs to a new type of fructose-1,6-bisphosphatase, while the latter is a typical enzyme similar to the cytosolic and chloroplastic forms from eukaryotic cells [Tamoi et al., Arch. Biochem. Biophys., 334, 1996, 27-36]. The genes of F-I and F-II were found in three species of cyanobacteria, Synechocystis PCC 6803, Anabaena 7120, and Plectonema boryanum according to the results of Southern hybridization with a probe from the S. 7942 F-I and F-II genes. In Western blotting, antibody raised against the S. 7942 F-I cross-reacted with a protein band corresponding to the F-I in each crude extract from cyanobacterial cells, whereas the antibody against F-II failed to cross-react with any protein band corresponding to the F-II. In cyanobacterial cells, only one form of F-I has been resolved by ion-exchange chromatography at same concentration of NaCl as shown in the F-I of S. 7942. The F-I from Synechocystis 6803 has been purified to electrophoretic homogeneity. The enzyme hydrolyzed both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate. The apparent K(m) values of the enzyme for fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were 57 +/- 2.4 and 180 +/- 6.3 microM, respectively. The enzyme activity was inhibited by AMP with a Ki value of 0.57 +/- 0.03 mM for fructose 1,6-bisphosphate and 0.35 +/- 0.02 mM for sedoheptulose 1,7-bisphosphate. The enzyme showed a molecular mass of 168 kDa which was composed of four identical subunits. The activities of FBPase and SBPase from the F-I were resistant to hydrogen peroxide up to 1 mM. The nucleotide sequence of the S. 6803 F-I gene showed an open reading frame of 1164 bp that encoded a protein of 388 amino acid residues (approx. molecular mass of 41.6 kDa). The deduced amino acid sequences had homologous sequences with the S. 7942 F-I.

Chitinase Gene Expression in Response to Environmental Stresses in Arabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

The expression levels of three chitinase genes in Arabidopsis thaliana, AtChiA (class III), AtChiB (class I), and AtChiV (class IV), were examined under various stress conditions by semi-quantitative RT-PCR. Under normal growth conditions, the AtChiB and AtChiV genes were expressed in most organs of Arabidopsis plants at all growth stages, whereas the AtChiA gene was not expressed at all. The class III AtChiA gene was expressed exclusively when the plants were exposed to environmental stresses, especially to salt and wound stresses. Treatment of Arabidopsis plants with allosamidin, which inhibits class III chitinases, did not affect the growth rate. Surprisingly, however, the plants treated with allosamidin were more tolerant of abiotic stresses (cold, freezing, heat, and strong light) than the control plants. It also appeared that allosamidin enhances AtChiA and AtChiB expression under heat and strong light stresses. Allosamidin is likely to enhance abiotic stress tolerance, probably through crosstalk between the two signaling pathways for biotic and abiotic stress responses.

AtNUDX6, an ADP-Ribose/NADH Pyrophosphohydrolase in Arabidopsis, Positively Regulates NPR1-Dependent Salicylic Acid Signaling

Here, we investigated the physiological role of Arabidopsis (Arabidopsis thaliana) AtNUDX6, the gene encoding ADP-ribose (Rib)/NADH pyrophosphohydrolase, using its overexpressor (Pro35S:AtNUDX6) or disruptant (KO-nudx6). The level of NADH in Pro35S:AtNUDX6 and KO-nudx6 plants was decreased and increased, respectively, compared with that of the control plants, while the level of ADP-Rib was not changed in either plant. The activity of pyrophosphohydrolase toward NADH was enhanced and reduced in the Pro35S:AtNUDX6 and KO-nudx6 plants, respectively. The decrease in the activity of NADH pyrophosphohydrolase and the increase in the level of NADH were observed in the rosette and cauline leaves, but not in the roots, of the KO-nudx6 plants. Notably, the expression level of AtNUDX6 and the activity of NADH pyrophosphohydrolase in the control plants, but not in the KO-nudx6 plants, were increased by the treatment with salicylic acid (SA). The expression of SA-induced genes (PR1, WRKY70, NIMIN1, and NIMIN2) depending on NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key component required for pathogen resistance, was significantly suppressed and enhanced in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively, under the treatment with SA. Induction of thioredoxin h5 (TRX-h5) expression, which catalyzes a SA-induced NPR1 activation, was suppressed and accelerated in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively. The expression of isochorismate synthase1, required for the regulation of SA synthesis through the NPR1-mediated feedback loop, was decreased and increased in the KO-nudx6 and Pro35S:AtNUDX6 plants, respectively. Judging from seed germination rates, the KO-nudx6 plants had enhanced sensitivity to the toxicity of high-level SA. These results indicated that AtNUDX6 is a modulator of NADH rather than ADP-Rib metabolism and that, through induction of TRX-h5 expression, AtNUDX6 significantly impacts the plant immune response as a positive regulator of NPR1-dependent SA signaling pathways.

Arabidopsis NADPH oxidases, AtrbohD and AtrbohF, are essential for jasmonic acid-induced expression of genes regulated by MYC2 transcription factor

To clarify genetically the involvement of two Arabidopsis NADPH oxidases (AtrbohD and AtrbohF) in the jasmonic acid (JA) signaling pathway, we characterized single knockout mutants lacking either Atrboh. The accumulation of reactive oxygen species (ROS) and expression of the genes regulated by MYC2, a transcription factor involved in the JA-evoked response, were significantly suppressed by treatment with methyl JA (MeJA) in both mutants. Further experiments using knockout mutants lacking CORONATINE-INSENSITIVE1 (COI1), a master regulator of the JA-evoked response, and MYC2 indicated a possibility that the production of ROS via Atrbohs depends on the function of COI1, but not MYC2.

NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803.

Here we isolated and characterized two genes (slr1171, slr1992) designated gpx‐1 and gpx‐2, respectively, encoding glutathione peroxidase (GPX)‐like proteins (Gpx‐1, Gpx‐2) from Synechocystis PCC 6803. The deduced amino acid sequences for gpx‐1 and gpx‐2 showed high similarity to those of GPX‐like proteins from higher plants and mammalian GPXs, respectively. Surprisingly, both recombinant proteins in Escherichia coli were able to utilize NADPH, but not reduced glutathione, as an electron donor and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides as an acceptor. It seems accurate to refer to Gpx‐1 and Gpx‐2 as NADPH‐dependent GPX‐like proteins that serve as a new defense system for the reduction of unsaturated fatty acid hydroperoxides.

The 26S Proteasome Function and Hsp90 Activity Involved in the Regulation of HsfA2 Expression in Response to Oxidative Stress

Heat shock transcription factor A2 (HsfA2) is induced under environmental stress and regulates transcription of various defense-related genes. Thus HsfA2 plays an important role in induction of defenses against different types of environmental stress, but its mode of regulation remains unknown. To clarify the signal transduction pathway involved in the regulation of HsfA2 expression, we investigated the effect of MG132, a 26S proteasome inhibitor, or geldanamycin (GDA), a heat shock protein 90 (Hsp90) inhibitor, on the transcription of HsfA2 and its targets, Hsp18.1-CI and ascorbate peroxidase 2 (Apx2), in Arabidopsis T87 cells. The levels of transcripts were significantly increased by treatment with MG132 or GDA. Overexpression of a dexamethazone-inducible dominant-negative form of Hsp90.2 in Arabidopsis plants caused significant expression of HsfA2 and its target gene on treatment with the compound. Treatment with MG132 or GDA had no effect on intracellular levels of reactive oxygen species (ROS). Interestingly, the levels of polyubiquitinated proteins as well as the levels of HsfA2 transcript were rapidly increased under oxidative stress derived from treatment with H2O2 or methylviologen, while they were completely suppressed by pre-treatment with ascorbate, a scavenger of ROS, under oxidative stress. The present findings suggest that the inhibition of 26S proteasome function and/or Hsp90 activity is involved in the induction of HsfA2 expression in response to oxidative stress.