Yukinori Yabuta

Galactinol and Raffinose Constitute a Novel Function to Protect Plants from Oxidative Damage

Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides that function as osmoprotectants in plant cells. In leaves of Arabidopsis (Arabidopsis thaliana) plants overexpressing heat shock transcription factor A2 (HsfA2), the transcription of GolS1, -2, and -4 and raffinose synthase 2 (RS2) was highly induced; thus, levels of galactinol and raffinose increased compared with those in wild-type plants under control growth conditions. In leaves of the wild-type plants, treatment with 50 μm methylviologen (MV) increased the transcript levels of not only HsfA2, but also GolS1, -2, -3, -4, and -8 and RS2, -4, -5, and -6, the total activities of GolS isoenzymes, and the levels of galactinol and raffinose. GolS1- or GolS2-overexpressing Arabidopsis plants (Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29) had increased levels of galactinol and raffinose in the leaves compared with wild-type plants under control growth conditions. High intracellular levels of galactinol and raffinose in the transgenic plants were correlated with increased tolerance to MV treatment and salinity or chilling stress. Galactinol and raffinose effectively protected salicylate from attack by hydroxyl radicals in vitro. These findings suggest the possibility that galactinol and raffinose scavenge hydroxyl radicals as a novel function to protect plant cells from oxidative damage caused by MV treatment, salinity, or chilling.

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.

Hydroperoxide reduction by thioredoxin‐specific glutathione peroxidase isoenzymes of Arabidopsis thaliana

Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1–8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, ‐2, ‐5 and ‐6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2–7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2–4.0 and 14.0–25.4 µm, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.

Arabidopsis NAC Transcription Factor, ANAC078, Regulates Flavonoid Biosynthesis under High-light

We have isolated a combination of high-light and heat-shock (HL + HS) stress-inducible genes, including a NAC transcription factor designated ANAC078. Here we explored the physiological function of ANAC078 under HL stress. Yeast transcription activity assays showed that ANAC078 functions as a transcriptional activator. A fusion protein composed of green fluorescent protein (GFP) and full-length ANAC078 was detected in the nucleus and cytoplasm, while fusion proteins comprising GFP and ANAC078 deleted of a putative transmembrane motif were found only in the nucleus. In ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078-43), the transcription of 166 genes was up-regulated compared with the levels in wild-type plants under HL (1200 micromol m(-2) s(-1), 30 degrees C). These genes included some for transcription factors regulating the expression of genes related to the biosynthesis of flavonoids. Interestingly, the transcript levels of some genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in Ox-ANAC078 plants and reduced in knockout ANAC078 plants (KO-ANAC078) compared with wild-type plants under HL stress. The present findings suggest that ANAC078 protein is associated with the induction of genes related to flavonoid biosynthesis, leading to the accumulation of anthocyanins, in response to HL stress.

A Bacterial Transgene for Catalase Protects Translation of D1 Protein during Exposure of Salt-Stressed Tobacco Leaves to Strong Light

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum ‘Xanthi’) and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [35S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplasts translational machinery to salt stress by scavenging hydrogen peroxide.

The contribution of carbohydrates including raffinose family oligosaccharides and sugar alcohols to protection of plant cells from oxidative damage

Recently we have reported that high intracellular levels of galactinol and raffinose in Arabidopsis plants overexpressing the heat shock transcription factor A2 or galactinol synthase are correlated with increased tolerance to methylviologen treatment and salinity or chilling stress, and galactinol and raffinose also are found to effectively protect salicylate from attack by hydroxyl radicals in vitro. These findings indicate that galactinol and raffinose act not only as osmoprotectants, but also as antioxidants in the leaves of Arabidopsis plants. At the same time, we found that the rate constant (1.1 ± 0.29 x 1010 M-1s-1) for the reaction between stachyose and hydroxyl radicals was higher than those of galactinol and raffinose and typical antioxidants. The accumulation of stachyose was only observed in seeds. Furthermore, glucose, fructose, and sucrose which are abundant in higher plants efficiently scavenge hydroxyl radicals. Judging from the radical scavenging activity and the intracellular level of each compound reported here and previously, we suggest that carbohydrates including raffinose family oligosaccharides and sugar alcohols are present at high levels under normal and/or stressfull conditions, and act as antioxidants to protect plant cells from oxidative damage and maintain redox homeostasis. Addendum to: Nishizawa A, Yabuta Y, Shigeoka S. Galactinol and raffinose constitute a novel function to protect plants from oxidative damage. Plant Physiol 2008; 147:1251-63.

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.

The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/l-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of l-galactonate and l-galactono-1,4-lactone with zinc ion as a cofactor. The apparent Km values for l-galactonate and l-galactono-1,4-lactone were 1.55 ± 0.3 and 1.67 ± 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with l-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/l-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

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.