Publications by authors named "Takanori Maruta"

55 Publications

The D-mannose/L-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physicomitrium patens.

Plant J 2021 Jul 10. Epub 2021 Jul 10.

Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the D-mannose/L-galactose (D-Man/L-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophyte, while the D-galacturonate (D-GalUA) pathway is distributed in parasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the D-Man/L-Gal pathway and an orthologous gene encoding aldonolactonae (ALase) - a key enzyme for the D-GalUA pathway - Physicomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. P. patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of L-galactonic acid to the final precursor L-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encodes the rate-limiting enzyme GDP-L-Gal phosphorylase in the D-Man/L-Gal pathway, markedly was decreased to 46% and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthestic pathway in P.patens is the D-Man/L-Gal pathway, not the D-GalUA pathway and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.
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http://dx.doi.org/10.1111/tpj.15413DOI Listing
July 2021

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo-oxidative stress.

Plant J 2021 May 24. Epub 2021 May 24.

Graduate School of Natural Science and Technology, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

High-light (HL) stress enhances the production of H O from the photosynthetic electron transport chain in chloroplasts, potentially causing photo-oxidative damage. Although stromal and thylakoid membrane-bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H O -scavenging enzymes in chloroplasts, their knockout mutants do not exhibit a visible phenotype under HL stress. Trans-thylakoid proton gradient (∆pH)-dependent mechanisms exist for controlling H O production from photosynthesis, such as thermal dissipation of light energy and downregulation of electron transfer between photosystems II and I, and these may compensate for the lack of APXs. To test this hypothesis, we focused on a proton gradient regulation 5 (pgr5) mutant, wherein both ∆pH-dependent mechanisms are impaired, and an Arabidopsis sapx tapx double mutant was crossed with the pgr5 single mutant. The sapx tapx pgr5 triple mutant exhibited extreme sensitivity to HL compared with its parental lines. This phenotype was consistent with cellular redox perturbations and enhanced expression of many oxidative stress-responsive genes. These findings demonstrate that the PGR5-dependent mechanisms compensate for chloroplast APXs, and vice versa. An intriguing finding was that the failure of induction of non-photochemical quenching in pgr5 (because of the limitation in ∆pH formation) was partially recovered in sapx tapx pgr5. Further genetic studies suggested that this recovery was dependent on the NADH dehydrogenase-like complex-dependent pathway for cyclic electron flow around photosystem I. Together with data from the sapx tapx npq4 mutant, we discuss the interrelationship between APXs and ∆pH-dependent mechanisms under HL stress.
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http://dx.doi.org/10.1111/tpj.15352DOI Listing
May 2021

GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation.

Nat Plants 2020 05 11;6(5):533-543. Epub 2020 May 11.

Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.
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http://dx.doi.org/10.1038/s41477-020-0645-zDOI Listing
May 2020

Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light-Induced Ascorbate Accumulation.

Plant Physiol 2020 05 23;183(1):112-122. Epub 2020 Mar 23.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane 690-8504, Japan

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis () triple mutant lacking all three genes (herein called ∆) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated ∆ Arabidopsis plants as well as a quadruple mutant line combining ∆ with an additional mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the mutant that contains ∼30% of the wild-type GSH level. The mutant accumulated ascorbate at a wild-type level under high light; however, when the mutation was combined with ∆, there was near-complete inhibition of high-light-dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in ∆ plants, and that both DHAR activity and GSH content set a threshold for high-light-induced ascorbate accumulation.
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http://dx.doi.org/10.1104/pp.19.01556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210653PMC
May 2020

Comparative proteomic analysis of mitochondria isolated from Euglena gracilis under aerobic and hypoxic conditions.

PLoS One 2019 31;14(12):e0227226. Epub 2019 Dec 31.

Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, Matsue, Shimane, Japan.

The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77-0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938325PMC
April 2020

Chloroplast development activates the expression of ascorbate biosynthesis-associated genes in Arabidopsis roots.

Plant Sci 2019 Jul 16;284:185-191. Epub 2019 Apr 16.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan; Graduate School of Natural Science and Technology, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan; Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Transcriptional activation of ascorbate biosynthesis-associated genes under illumination is one of the important steps in ascorbate pool size regulation in photosynthetic tissues. Several biological processes within chloroplasts such as photosynthesis are required for this activation, suggesting functional chloroplasts to play a key role. We herein found that when grown on agar plate, ascorbate content in Arabidopsis non-photosynthetic tissues, roots, are unexpectedly almost comparable to that in shoots. The high accumulation of ascorbate was particularly observed in root regions closer to the root-hypocotyl junction, in which chloroplast development occurred because of a direct exposure to light. When chloroplast development in roots were further stimulated by shoot removal, the expression of biosynthetic genes, especially VTC2 gene that encodes GDP-l-galactose phosphorylase, was activated, resulting in an increase in ascorbate pool size. These positive effects were canceled when the roots were treated with a photosynthetic inhibitor. A null mutation in the LONG HYPOCOTYL 5 (HY5) gene almost completely inhibited root greening as well as the VTC2 expression. Overall, these findings show that chloroplast development can trigger the expression of ascorbate biosynthesis-associated genes not only in leaves but also in roots.
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http://dx.doi.org/10.1016/j.plantsci.2019.04.012DOI Listing
July 2019

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

PLoS One 2019 16;14(1):e0210755. Epub 2019 Jan 16.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334954PMC
November 2019

Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis.

Sci Rep 2017 10 18;7(1):13504. Epub 2017 Oct 18.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan.

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.
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http://dx.doi.org/10.1038/s41598-017-14077-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647427PMC
October 2017

Biochemistry and Physiology of Reactive Oxygen Species in Euglena.

Adv Exp Med Biol 2017 ;979:47-64

Faculty of Agriculture, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are by-products of various metabolic processes in aerobic organisms including Euglena. Chloroplasts and mitochondria are the main sites of ROS generation by photosynthesis and respiration, respectively, through the active electron transport chain. An efficient antioxidant network is required to maintain intracellular ROS pools at optimal conditions for redox homeostasis. A comparison with the networks of plants and animals revealed that Euglena has acquired some aspects of ROS metabolic process. Euglena lacks catalase and a typical selenocysteine containing animal-type glutathione peroxidase for hydrogen peroxide scavenging, but contains enzymes involved in ascorbate-glutathione cycle solely in the cytosol. Ascorbate peroxidase in Euglena, which plays a central role in the ascorbate-glutathione cycle, forms a unique intra-molecular dimer structure that is related to the recognition of peroxides. We recently identified peroxiredoxin and NADPH-dependent thioredoxin reductase isoforms in cellular compartments including chloroplasts and mitochondria, indicating the physiological significance of the thioredoxin system in metabolism of ROS. Besides glutathione, Euglena contains the unusual thiol compound trypanothione, an unusual form of glutathione involving two molecules of glutathione joined by a spermidine linker, which has been identified in pathogenic protists such as Trypanosomatida and Schizopyrenida. Furthermore, in contrast to plants, photosynthesis by Euglena is not susceptible to hydrogen peroxide because of resistance of the Calvin cycle enzymes fructose-1,6-bisphosphatse, NADP-glyceraldehyde-3-phosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase to hydrogen peroxide. Consequently, these characteristics of Euglena appear to exemplify a strategy for survival and adaptation to various environmental conditions during the evolutionary process of euglenoids.
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http://dx.doi.org/10.1007/978-3-319-54910-1_4DOI Listing
October 2017

Glucan synthase-like 2 is indispensable for paramylon synthesis in Euglena gracilis.

FEBS Lett 2017 05 7;591(10):1360-1370. Epub 2017 May 7.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.

The phytoflagellate Euglena gracilis produces a large amount of paramylon (PM), a conglomerate of liner β-1,3-glucan chains, as a storage polysaccharide. PM is synthesized from uridine diphosphate-glucose, but its mechanism of formation is largely unknown. Two enzymes, glucan synthase-like (EgGSL) 1 and EgGSL2 were previously identified as candidates for PM synthesis in a Euglena transcriptome analysis. Here, we performed a reverse genetic analysis on these enzymes. Knockdown of EgGSL2, but not EgGSL1, significantly inhibits PM accumulation in Euglena cells. Additionally, β-1,3-glucan synthesis is detected in a PM-associated membrane fraction extracted from Euglena cells. Our findings indicate that EgGSL2 is the predominant enzyme for PM biosynthesis.
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http://dx.doi.org/10.1002/1873-3468.12659DOI Listing
May 2017

Loss-of-function of an Arabidopsis NADPH pyrophosphohydrolase, AtNUDX19, impacts on the pyridine nucleotides status and confers photooxidative stress tolerance.

Sci Rep 2016 11 22;6:37432. Epub 2016 Nov 22.

Department of Advanced Bioscience, Faculty of Agriculture, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan.

The levels and redox states of pyridine nucleotides, such as NADP(H), regulate the cellular redox homeostasis, which is crucial for photooxidative stress response in plants. However, how they are controlled is poorly understood. An Arabidopsis Nudix hydrolase, AtNUDX19, was previously identified to have NADPH hydrolytic activity in vitro, suggesting this enzyme to be a regulator of the NADPH status. We herein examined the physiological role of AtNUDX19 using its loss-of-function mutants. NADPH levels were increased in nudx19 mutants under both normal and high light conditions, while NADP and NAD levels were decreased. Despite the high redox states of NADP(H), nudx19 mutants exhibited high tolerance to moderate light- or methylviologen-induced photooxidative stresses. This tolerance might be partially attributed to the activation of either or both photosynthesis and the antioxidant system. Furthermore, a microarray analysis suggested the role of ANUDX19 in regulation of the salicylic acid (SA) response in a negative manner. Indeed, nudx19 mutants accumulated SA and showed high sensitivity to the hormone. Our findings demonstrate that ANUDX19 acts as an NADPH pyrophosphohydrolase to modulate cellular levels and redox states of pyridine nucleotides and fine-tunes photooxidative stress response through the regulation of photosynthesis, antioxidant system, and possibly hormonal signaling.
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http://dx.doi.org/10.1038/srep37432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118724PMC
November 2016

Arabidopsis clade IV TGA transcription factors, TGA10 and TGA9, are involved in ROS-mediated responses to bacterial PAMP flg22.

Plant Sci 2016 Nov 28;252:12-21. Epub 2016 Jun 28.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan. Electronic address:

Reactive oxygen species (ROS) produced in chloroplasts have been proposed to act as signaling molecules for plant immunity through pathogen-associated molecular patterns (PAMPs), such as flg22. To elucidate this process, we herein conducted genetic screening of flg22-sensitive mutants from T-DNA insertion lines lacking chloroplastic HO-responsive genes. The results obtained showed that knockout mutants lacking a clade IV TGA transcription factor, TGA10, were more sensitive to the flg22 treatment than wild-type plants. Furthermore, although no flg22-sensitive phenotype was detected in the knockout mutant of another clade IV TGA9, double knockout tga9 tga10 mutants showed more sensitivity to flg22 than single knockout mutants. Transcripts of TGA10 and TGA9 were strongly induced by flg22 in leaves, and this was facilitated by the double knockout of stromal and thylakoid-bound ascorbate peroxidases (APX), which are major HO scavengers in chloroplasts. The flg22-induced HO accumulation was maintained at high level in these APXs mutants, indicating the clade IV TGAs may be induced by the ROS. Furthermore, TGA10 was required for the complete activation of the expression of several flg22-responsive genes in plants treated with this PAMP. These have provided a new insight into the relationship between the TGA transcription factors and ROS-mediated signaling in PAMPs responses.
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http://dx.doi.org/10.1016/j.plantsci.2016.06.019DOI Listing
November 2016

De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions.

BMC Genomics 2016 Mar 3;17:182. Epub 2016 Mar 3.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0052, Japan.

Background: The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions.

Results: The E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel β-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment.

Conclusion: The RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.
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http://dx.doi.org/10.1186/s12864-016-2540-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778363PMC
March 2016

Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis.

Biosci Biotechnol Biochem 2016 May 1;80(5):870-7. Epub 2016 Mar 1.

a Faculty of Agriculture, Department of Advanced Bioscience , Kinki University , Nara , Japan.

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.
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http://dx.doi.org/10.1080/09168451.2015.1135042DOI Listing
May 2016

Diversity and Evolution of Ascorbate Peroxidase Functions in Chloroplasts: More Than Just a Classical Antioxidant Enzyme?

Plant Cell Physiol 2016 Jul 6;57(7):1377-1386. Epub 2016 Jan 6.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504 Japan

Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of ascorbate peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of HO Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to HO A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to HO may have been acquired during plant evolution, thereby allowing for the flexible use of HO as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic HO as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.
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http://dx.doi.org/10.1093/pcp/pcv203DOI Listing
July 2016

Enhancement of photosynthetic capacity in Euglena gracilis by expression of cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase leads to increases in biomass and wax ester production.

Biotechnol Biofuels 2015 30;8:80. Epub 2015 May 30.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan ; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, 102-0076 Japan.

Background: Microalgae have recently been attracting attention as a potential platform for the production of biofuels. Euglena gracilis, a unicellular phytoflagellate, has been proposed as an attractive feedstock to produce biodiesel because it can produce large amounts of wax esters, consisting of medium-chain fatty acids and alcohols with 14:0 carbon chains. E. gracilis cells highly accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. When grown aerobically and then transferred into anaerobic conditions, E. gracilis cells degrade paramylon to actively synthesize and accumulate wax esters. Thus, the enhanced accumulation of paramylon through the genetic engineering of photosynthesis should increase the capacity for wax ester production.

Results: We herein generated transgenic Euglena (EpFS) cells expressing the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), which is involved in the Calvin cycle, to enhance its photosynthetic activity. FBP/SBPase was successfully expressed within Euglena chloroplasts. The cell volume of the EpFS4 cell line was significantly larger than that of wild-type cells under normal growth conditions. The photosynthetic activity of EpFS4 cells was significantly higher than that of wild type under high light and high CO2, resulting in enhanced biomass production, and the accumulation of paramylon was increased in transgenic cell lines than in wild-type cells. Furthermore, when EpFS cell lines grown under high light and high CO2 were placed on anaerobiosis, the productivity of wax esters was approximately 13- to 100-fold higher in EpFS cell lines than in wild-type cells.

Conclusion: Our results obtained here indicate that the efficiency of biomass production in E. gracilis can be improved by genetically modulating photosynthetic capacity, resulting in the enhanced production of wax esters. This is the first step toward the utilization of E. gracilis as a sustainable source for biofuel production under photoautotrophic cultivation.
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http://dx.doi.org/10.1186/s13068-015-0264-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459067PMC
June 2015

Identification and characterization of Arabidopsis AtNUDX9 as a GDP-d-mannose pyrophosphohydrolase: its involvement in root growth inhibition in response to ammonium.

J Exp Bot 2015 Sep 6;66(19):5797-808. Epub 2015 Jun 6.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

GDP-d-mannose (GDP-d-Man) is an important intermediate in ascorbic acid (AsA) synthesis, cell wall synthesis, protein N-glycosylation, and glycosylphosphatidylinositol-anchoring in plants. Thus, the modulation of intracellular levels of GDP-d-Man could be important for maintaining various cellular processes. Here an Arabidopsis GDP-d-Man pyrophosphohydrolase, AtNUDX9 (AtNUDT9; At3g46200), which hydrolysed GDP-d-Man to GMP and mannose 1-phosphate, was identified. The K m and V max values for GDP-d-Man of AtNUDX9 were 376±24 μM and 1.61±0.15 μmol min(-1) mg(-1) protein, respectively. Among various tissues, the expression levels of AtNUDX9 and the total activity of GDP-d-Man pyrophosphohydrolase were the highest in the roots. The GDP-d-Man pyrophosphohydrolase activity was increased in the root of plants grown in the presence of ammonium. No difference was observed in the levels of AsA in the leaf and root tissues of the wild-type and knockout-nudx9 (KO-nudx9) plants, whereas a marked increase in N-glycoprotein levels and enhanced growth were detected in the roots of KO-nudx9 plants in the presence of ammonium. These results suggest that AtNUDX9 is involved in the regulation of GDP-d-Man levels affecting ammonium sensitivity via modulation of protein N-glycosylation in the roots.
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http://dx.doi.org/10.1093/jxb/erv281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566977PMC
September 2015

Biochemical and physiological analyses of NADPH-dependent thioredoxin reductase isozymes in Euglena gracilis.

Plant Sci 2015 Jul 30;236:29-36. Epub 2015 Mar 30.

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo 102-0076, Japan. Electronic address:

At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.
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http://dx.doi.org/10.1016/j.plantsci.2015.03.016DOI Listing
July 2015

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

New Phytol 2015 May 27;206(3):1013-1023. Epub 2015 Jan 27.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara, 631-8505, Japan.

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.
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http://dx.doi.org/10.1111/nph.13309DOI Listing
May 2015

Transcriptional control of vitamin C defective 2 and tocopherol cyclase genes by light and plastid-derived signals: the partial involvement of GENOMES UNCOUPLED 1.

Plant Sci 2015 Feb 27;231:20-9. Epub 2014 Nov 27.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Electronic address:

Previous findings have suggested that light and plastid-derived signals are involved in the regulation of biosynthetic pathways for l-ascorbic acid (AsA) and tocopherols (Toc). Photosynthetic electron transport (PET) activity, plastid gene expression (PGE), and the tetrapyrrole metabolism have been identified as signals that regulate nuclear gene expression through the GENOMES UNCOUPLED 1 (GUN1) protein. Here, we examined the effects of disrupting GUN1 on these pathways. The expression of vitamin C defective 2 (VTC2) and tocopherol cyclase (TC) genes, which encode key enzymes in the AsA and Toc biosynthetic pathways, respectively, was affected by illumination and darkness in parallel with the levels of both these antioxidants. However, the GUN1 disruption had no effect on these biosynthetic pathways under light-dark conditions. All treatments that inhibited PET, PGE, and the tetrapyrrole metabolism interrupted both biosynthetic pathways; however, this was partially mitigated by the GUN1 disruption. The expression patterns of VTC2 and TC reflected the levels of both antioxidants under most of the conditions examined. Our results suggest that the transcriptional control of VTC2 and TC by light and plastid-derived signals is important for the regulation of the biosynthetic pathways, and that GUN1 is at least partially involved in the plastid-derived signals-dependent regulation.
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http://dx.doi.org/10.1016/j.plantsci.2014.11.007DOI Listing
February 2015

Cellular redox regulation, signaling, and stress response in plants.

Biosci Biotechnol Biochem 2014 ;78(9):1457-70

a Faculty of Agriculture, Department of Advanced Bioscience , Kinki University , Nara , Japan.

Cellular and organellar redox states, which are characterized by the balance between oxidant and antioxidant pool sizes, play signaling roles in the regulation of gene expression and protein function in a wide variety of plant physiological processes including stress acclimation. Reactive oxygen species (ROS) and ascorbic acid (AsA) are the most abundant oxidants and antioxidants, respectively, in plant cells; therefore, the metabolism of these redox compounds must be strictly and spatiotemporally controlled. In this review, we provided an overview of our previous studies as well as recent advances in (1) the molecular mechanisms and regulation of AsA biosynthesis, (2) the molecular and genetic properties of ascorbate peroxidases, and (3) stress acclimation via ROS-derived oxidative/redox signaling pathways, and discussed future perspectives in this field.
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http://dx.doi.org/10.1080/09168451.2014.942254DOI Listing
May 2015

Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

Biosci Biotechnol Biochem 2014 21;78(4):593-601. Epub 2014 May 21.

a Faculty of Life and Environmental Science, Department of Life Science and Biotechnology , Shimane University , Matsue , Japan.

Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.
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http://dx.doi.org/10.1080/09168451.2014.890037DOI Listing
August 2015

Transient expression analysis revealed the importance of VTC2 expression level in light/dark regulation of ascorbate biosynthesis in Arabidopsis.

Biosci Biotechnol Biochem 2014 16;78(1):60-6. Epub 2014 Apr 16.

a Department of Food and Nutritional Science, College of Bioscience and Biotechnology , Chubu University , Kasugai , Japan.

Ascorbate (AsA) is an important antioxidant and an enzyme cofactor involved in various metabolic pathways. In this study, we investigated the effects of estrogen (ES)-inducible transient expression of genes encoding enzymes involved in the d-mannose/l-galactose (d-Man/l-Gal) pathway for plant AsA biosynthesis on AsA levels under light and dark conditions. No significant difference was observed in AsA levels between Arabidopsis plants transiently expressing phosphomannose isomerase (PMI1), GDP-d-Man pyrophosphorylase (GMP/VTC1), GDP-Man-3',5'-epimerase (GME), and l-Gal 1-phosphate phosphatase (GPP/VTC4), but AsA levels in the plants transiently expressing GDP-l-Gal phosphorylase (GGP/VTC2) were 2.5-fold higher than those in control plants 7 d after ES treatment. The increase in AsA levels under continuous light conditions and the decrease in AsA levels under dark conditions were enhanced and suppressed, respectively, in the ES-treated plants. These results suggest that GGP/VTC2 acts as a rate-limiting step regulating AsA biosynthesis in response to light and dark conditions.
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http://dx.doi.org/10.1080/09168451.2014.877831DOI Listing
March 2015

Ferulic acid 5-hydroxylase 1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis.

Plant Sci 2014 Apr 28;219-220:61-8. Epub 2014 Jan 28.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Electronic address:

Anthocyanins are important for preventing photoinhibition and photodamage. By comprehensive reverse genetic analysis of chloroplast-produced H2O2-responsive genes, we isolated here an anthocyanin-deficient mutant under photooxidative stress, which lacked ferulate 5-hydroxylase 1 (FAH1) involved in the phenylpropanoid pathway. Interestingly, the expression of anthocyanin biosynthesis-associated genes was also inhibited in this mutant. These findings suggest that FAH1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis. Furthermore, we found that estrogen-inducible silencing of thylakoid membrane-bound ascorbate peroxidase, which is a major H2O2-scavenging enzyme in chloroplasts, enhances the expression of FAH1 and anthocyanin biosynthesis-associated genes and accumulation of anthocyanin without any application of stress. Thus, it is likely that chloroplastic H2O2 activates FAH1 expression to induce anthocyanin accumulation for protecting cells from photooxidative stress.
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http://dx.doi.org/10.1016/j.plantsci.2014.01.003DOI Listing
April 2014

Activation of γ-aminobutyrate production by chloroplastic H(2)O(2) is associated with the oxidative stress response.

Biosci Biotechnol Biochem 2013 7;77(2):422-5. Epub 2013 Feb 7.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan.

We isolated an Arabidopsis knockout line lacking glutamate decarboxylase 1 (GAD1), one that produced γ-aminobutyrate (GABA), as an oxidative stress-insensitive mutant, and found that chloroplastic H(2)O(2) enhances GAD1 expression and GABA levels. This suggests a possible relationship between GABA metabolism and the chloroplastic H(2)O(2)-mediated stress response.
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http://dx.doi.org/10.1271/bbb.120825DOI Listing
August 2013

Enzymatic and molecular characterization of Arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26.

Biosci Biotechnol Biochem 2012 7;76(12):2236-41. Epub 2012 Dec 7.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nara, Japan.

Not only in bacteria but also in plant cells, guanosine-3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrolases, AtNUDX11, 15, 25, and 26, having ppGpp pyrophosphohydrolase activity from Arabidopsis plants. Among these, AtNUDX26 localized in chloroplasts had the highest Vmax and kcat values, leading to high catalytic efficiency, kcat/Km. The activity of AtNUDX26 required Mg2+ or Mn2+ ions as cofactor and was optimal at pH 9.0 and 50 °C. The expression of AtNUDX26 and of ppGpp metabolism-associated genes was regulated by various types of stress, suggesting that AtNUDX26 regulates cellular ppGpp levels in response to stress and impacts gene expression in chloroplasts. This is the first report on the molecular properties of ppGpp pyrophosphohydrolases in plants.
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http://dx.doi.org/10.1271/bbb.120523DOI Listing
June 2013

Subcellular and subnuclear distribution of high-light responsive serine/arginine-rich proteins, atSR45a and atSR30, in Arabidopsis thaliana.

Biosci Biotechnol Biochem 2012 7;76(11):2075-81. Epub 2012 Nov 7.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.

Here, we demonstrated the involvement of the domains in Arabidopsis high-light responsive serine/arginine-rich (SR) and SR-like proteins, atSR30 and atSR45a, respectively, in subcellular and subnuclear distribution using a series of structural domain-deleted mutants. Judging from the localization of the transiently expressed domain-deleted mutants in onion epidermal cells, the C terminal low complexity domain rich in arginine-serine repeats (C-RS) domain of atSR30 appeared to be necessary for the nuclear localization. On the other hand, the N-terminal RS (N-RS) domain of atSR45a was necessary for the accurate nuclear localization, although the N- or C-RS domain alone was sufficient for the nuclear speckled organization. The phosphorylation of RS domains of atSR45a is irrelevant to the regulation of its localization. atSR45a and atSR30 were co-localized in the speckles, suggesting their collaborative roles in the regulation of alternative splicing events.
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http://dx.doi.org/10.1271/bbb.120425DOI Listing
May 2013

Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering.

Transgenic Res 2013 Apr 19;22(2):391-402. Epub 2012 Sep 19.

School of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, 4-101 Koyama-minami, Tottori, 680-8550, Japan.

Vitamin E (tocopherol: Toc) is an important lipid-soluble antioxidant synthesized in chloroplasts. Among the 8 isoforms of vitamin E, α-Toc has the highest activity in humans. To generate transgenic plants with enhanced vitamin E activity, we applied a chloroplast transformation technique. Three types of the transplastomic tobacco plants (pTTC, pTTMT and pTTC-TMT) carrying the Toc cyclase (TC) or γ-Toc methyltransferase (γ-TMT) gene and the TC plus γ-TMT genes as an operon in the plastid genome, respectively, were generated. There was a significant increase in total levels of Toc due to an increase in γ-Toc in the pTTC plants. Compared to the wild-type plants, Toc composition was altered in the pTTMT plants. In the pTTC-TMT plants, total Toc levels increased and α-Toc was a major Toc isoform. Furthermore, to use chloroplast transformation to produce α-Toc-rich vegetable, TC-overexpressing transplastomic lettuce plants (pLTC) were generated. Total Toc levels and vitamin E activity increased in the pLTC plants compared with the wild-type lettuce plants. These findings indicated that chloroplast genetic engineering is useful to improve vitamin E quality and quantity in plants.
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http://dx.doi.org/10.1007/s11248-012-9656-5DOI Listing
April 2013

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H(2)O(2) in Arabidopsis plants.

Biochim Biophys Acta 2012 Dec 18;1820(12):1901-7. Epub 2012 Aug 18.

Department of Advanced Bioscience, Kinki University, Nara, Japan.

Background: Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods: We studied the effect of wounding and MeJA treatment on the levels of H(2)O(2) and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results: The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H(2)O(2) accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H(2)O(2) were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General Significance: This is the first report demonstrating that H(2)O(2)-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.
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http://dx.doi.org/10.1016/j.bbagen.2012.08.003DOI Listing
December 2012

Relationship between chloroplastic H 2O 2 and the salicylic acid response.

Plant Signal Behav 2012 Aug 27;7(8):944-6. Epub 2012 Jul 27.

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan.

Reactive oxygen species (ROS) act as signaling molecules for regulating plant responses to abiotic and biotic stress and there exist source- and kind-specific pathways for ROS signaling. Recently, we created a novel system for producing H 2O 2 in Arabidopsis chloroplasts by chemical-dependent thylakoid membrane-bound ascorbate peroxidase (tAPX) silencing using an estrogen-inducible RNAi method. Microarray analysis revealed that the expression of a large set of genes was altered in response to tAPX silencing, some of which are known to be involved in pathogen response/resistance. Furthermore, we found that tAPX silencing enhances the levels of salicylic acid (SA) and the response to SA, a central regulator for biotic stress response. In this addendum, we describe the relationship between chloroplastic H 2O 2 and SA in stress response, and discuss the function of the kind- and source-specific ROS signaling in SA-mediated stress response.
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http://dx.doi.org/10.4161/psb.20906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474691PMC
August 2012
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