Akio Morita

Induction of suberin and increase of lignin content by excess boron in tobacco cells

Abstract Suspension-cultured tobacco cells (Nicotiana tabacum L. cv. Barley 21) were treated with 0.1 (control condition), 10, and 20 mm of boron (B). The histochemical observations revealed the deposition of suberin in the walls of the 10 and 20 mm B-treated cells. Lignin content and the amount of phenolics esterified to the polysaccharides of the walls of the 10 and 20 mm B-treated cells were also higher than those in the 0.1 mm of B. Lignosuberization of the 10 and 20 mm B-treated tobacco cells was accompanied by the increase in the activities of phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), peroxidase (PO), and polyphenol oxidase (PPO). In spite of the lignosuberization of tobacco cells after treatment with 10 and 20 mm of B, no significant reduction was observed in their viability, compared to those under the control conditions.

Characterization of AtSTOP1 Orthologous Genes in Tobacco and Other Plant Species

Diverse land plant species possess similar proteins that function in transcriptional regulation of aluminum tolerance. Aluminum (Al) and proton (H+) tolerances are essential traits for plants to adapt to acid soil environments. In Arabidopsis (Arabidopsis thaliana), these tolerances are mediated by a zinc-finger transcription factor, SENSITIVE TO PROTON RHIZOTOXICITY1 (AtSTOP1), which regulates the transcription of multiple genes critical for tolerance to both stressors. Here, the functions of orthologous proteins (STOP1-like proteins) in other plant species were characterized by reverse genetics analyses and in planta complementation assays. RNA interference of a gene for NtSTOP1 repressed Al and H+ tolerances of tobacco (Nicotiana tabacum) roots. Tobacco roots released citrate in response to Al, concomitant with the up-regulated transcription of an ortholog of an Al tolerance gene encoding a citrate-transporting multidrug and toxic compound extrusion protein. The RNA interference repression of NtSTOP1 blocked this process and also repressed the transcription of another orthologous gene for Al tolerance, ALUMINUM SENSITIVE3, which encodes a prokaryote-type transporter. These results demonstrated that NtSTOP1 regulates Al tolerance in tobacco through the transcriptional regulation of these genes. The in planta complementation assays revealed that other plant species, including woody plants, a legume, and a moss (Physcomitrella patens), possess functional STOP1-like proteins that can activate several H+ and Al-tolerance genes in Arabidopsis. Knocking out the gene encoding the STOP1-like protein decreased the Al tolerance of P. patens. Together, our results strongly suggest that transcriptional regulation by STOP1-like proteins is evolutionarily conserved among land plants and that it confers the ability to survive in acid soils through the transcriptional regulation of Al- and H+-tolerance genes.

δ13C values of organic carbon in cropland and forest soils in Japan

Abstract The carbon content and natural 13C abundance (δ13C) of soil organic carbon were determined for Japanese cropland and nearby forest soils by continuous flow CN analysis with mass spectrometry. The δ13C values of organic carbon from cropland soils in the Hokkaido District ranged between −27 and −24‰, suggesting that soil organic carbon originated mainly from C3 plants, while the δ13C values from those in Main Island, Shikoku, and Kyushu Districts ranged between −25 and −17‰, indicating that up to 70% of soil organic carbon originated from C4 plants (presumably Miscanthus sinensis). The δ13C values of organic carbon from Imaichi and Tanegashima forest soils showed that carbon was largely of C3 plant origin in the surface soils but in deeper horizons the values were strongly affected by the organic carbon of C4 plant origin. Sugarcane cultivation for 36 y in Tanegashima cropland soils had resulted in the increase of the soil δ13C values to around −15‰. In Shizuoka Prefecture, the δ13C values of soil organic carbon of non-volcanic ash Andosols under forests at Iwata and Makinohara were within the ranges of C3 plant origin. The values in volcanic ash Andosols at Fuji indicated that the soil carbon in the surface horizons mainly originated from C3 plants while that in deep soil horizons largely from C4 plants. Tea cultivation for more than 40 y had led to a decrease of the δ13C values at Fuji by 2-4‰. In the Miyako Island sugarcane fields, the δ13C values of the plow layers were apparently affected by sugarcane, a C4 plant species, compared to the subsoils where a large fraction of carbon originated from C3 forest plants. The shift from forest to sugarcane cultivation markedly decreased the carbon content and increased the δ13C values of soil organic carbon from −27-−25‰ to −17-−14‰ within 25 y.

Tea plant (Camellia sinensis L.) roots secrete oxalic acid and caffeine into medium containing aluminum

We examined the response of the tea plant (Camellia sinensis L.) to aluminum (Al) exposure under sterile conditions, focusing specifically on the secretion of low molecular mass organic compounds from roots. After germination in agar medium, tea seedlings together with medium were placed on agar containing 0.4 mM Al with 0.2% hematoxyline (hematoxylin-Al medium). The purple color of the hematoxylin-Al medium was observed to fade gradually, until none of the color remained 6 days later. The tea seedlings were then treated with simple calcium solution (0.2 mM, at pH 4.2) containing AlCl3, which ranged in concentration from 0 to 0.8 mM, for 24 hrs. The amount of oxalate secreted into the medium increased as the external Al concentration increased, while the concentrations of malate and citrate in the medium remained unchanged. Oxalate secretion started within 30 min after Al exposure and increased linearly thereafter. The findings demonstrated that oxalate was a key compound in the Al-tolerance mechanism employed by the tea plant, which detoxifies Al3+ externally in the rhizosphere. In addition to oxalate, caffeine was also secreted by tea roots in response to Al exposure. It is possible that caffeine excretion from the roots of tea plants may stimulate root growth through the inhibition of callose deposition in root tips.

Disclosure of the Fairy of Fairy-Ring-Forming Fungus Lepista sordida

Since the first scientific article about “fairy rings” in 1675 and subsequent studies reviewed in 1884, this phenomenon has been a mystery attributed to “fairies”. The tendency of all fungi to grow outward from the point of germination of the spore results in circular colonies in a widely varying group of fungi. Fairy rings are zones of stimulated grass growth. They appear as more or less continuous, circular bands of turfgrass that are darker green and faster growing than adjacent plants of the same species (Figure 1 A). These belts of greener plants can range from 10 to 30 cm wide, and the diameter of the circles they form is generally between 0.9 and 3.7 m. Fungi are responsible for this growth stimulation; presumably through the saprophytic action of the fungus mycelium, the protein portion of nonliving organic matter in the soil is decomposed to ammonia. The ammonia combines with other compounds, or is used as a substrate by successive bacteria to generate nitrites and nitrates. The resulting accumulation of nitrogen in the soil in a form readily available to higher plants causes the typical growth pattern of conspicuous bands of taller, darker green plants. 2] We questioned this accepted notion, even if it is partially true, and thus investigated the possibility of a specific plantgrowth-regulating substance(s) being produced by the fungi. We cultured a fairy-ring-forming fungus, Lepista sordida, examined the effect of the culture on the growth of turfgrass, and found that the culture supernatant promoted plant growth. L. sordida is widespread in northern temperate zones throughout the world, including the campus of our university where this study was conducted (Figure S1 in the Supporting Information). First, to confirm that our L. sordida strain exhibits growthpromoting activity, the cultivated mycelia were placed under bentgrass seedlings (Agrostis palustris) in a deep petri dish and incubated for three weeks. Grass treated with the fungus grew taller than untreated grass (Figure 1 B). Isolation of the active compound from the fungus was guided by its growth-regulating activity on bentgrass. The liquid-cultured fungus was filtered, the filtrate was fractionated by repeated chromatography, and the fractions were tested for their growth-regulating activity. This lead to the isolation and purification of the active compound, 2-azahypoxanthine (AHX; Figures 2 A, B, S2 and S3). Although AHX has been reported as a photolytic degradation product from an antitumor drug, dacarbazine (5-(3,3-dimethyltriazeno)imidazole-4-carboxamide) and has been synthesized, this is the first reported isolation of AHX from a natural source. AHX elongated the shoots and roots of bentgrass seedlings (Figure 2 B). The effect of AHX on shoots was observed at 0.2 and 1 mm and on roots at 0.05 and 0.2 mm. In order to confirm the presence of AHX in the interaction between the fungus and bentgrass (Figure 1 B and Figure S4), we analyzed the soil components by HPLC, H NMR spectroscopy and mass [a] Dr. J.-H. Choi, K. Fushimi, Prof. Dr. H. Kawagishi Graduate School of Science and Technology, Shizuoka University 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan) Fax: (+ 81) 54-238-4885 E-mail : achkawa@agr.shizuoka.ac.jp [b] N. Abe, H. Tanaka, Dr. S. Maeda, Prof. Dr. A. Morita, Prof. Dr. H. Kawagishi Department of Applied Biological Chemistry Faculty of Agriculture, Shizuoka University 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan) [c] M. Hara, Dr. R. Motohashi Department of Biological Science Faculty of Agriculture, Shizuoka University 836 Ohya, Suruga-ku, Shizuoka 422-8529 (Japan) [d] J. Matsunaga, Y. Eguchi, N. Ishigaki Suzuyo Research Institute, Ltd. 11-26, Tsukiji-cho, Shimizu-ku, Shizuoka 424-0944 (Japan) [e] Dr. D. Hashizume, Dr. H. Koshino RIKEN Hirosawa, Wako, Saitama 351-0198 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000112. Figure 1. Growth promotion of bentgrass by the fungus Lepista sordida. A) Fairy rings on the Turfgrass Study Field, Chiba Prefectural Agriculture and Forestry Research Center, Japan. B) Effect of the mycelia of L. sordida cultivated for three weeks on bentgrass. T1 and T2 represent bentgrass inoculated with 0.5 g and 1.5 g (fresh weight) of the mycelia, respectively.

Changes in peroxidase activity and lignin content of cultured tea cells in response to excess manganese

Abstract The present study was undertaken to identify the effects of manganese (Mn) on the activity of peroxidase (PO) and the amount of ascorbic acid (AsA) and lignin, as well as the cell viability of suspension-cultured tea cells (Camellia sinensis L. cv. Yabukita). Cells were grown in B5 medium (containing 0.06 mmol L−1 Mn) and cultured for 24 h in medium with a Mn concentration of 0.9 mmol L−1 as an excess treatment. No significant difference was observed between cellular growth and the viability of the cultured tea cells after treatment with excess Mn compared with cells grown under control conditions, although the content of Mn in cells in the excess Mn treatment was 12-fold higher than that of the control cells. The amount of total AsA was also not affected by the Mn treatment. The activity of ionically wall-bound peroxidase (IPO) increased in the presence of excess Mn, unlike the content of lignin. Conversely, the activities of soluble PO and covalently wall-bound PO decreased with excess Mn. These findings suggested that IPO might contribute to Mn tolerance in tea cells. However, its role in the mechanism(s) of Mn tolerance has not been elucidated.

Small Intestinal Goblet Cell Proliferation Induced by Ingestion of Soluble and Insoluble Dietary Fiber Is Characterized by An Increase in Sialylated Mucins in Rats

The study aimed to examine the effects of insoluble and soluble fibers on mucin sialylation and sulfation in the small intestine. First, diets containing soluble [konjac mannan (KM), psyllium, or guar gum; 50 g/kg) or insoluble (polystyrene foam, wheat bran, or cornhusk; 80 g/kg) fiber were fed to rats for 13 d. The fiber-fed groups had more goblet cells in the ileum than the fiber-free control group. High-iron diamine/alcian blue staining showed more sialylated mucin-producing cells in the fiber-fed groups than in the control, whereas sulfated mucin-producing cells were fewer (insoluble fibers) or unchanged (soluble fibers). Second, feeding KM (50 g/kg) and beet fiber (BF) (80 g/kg) diets for 7 d yielded a higher ileum Siat4C expression than the control, but Gal3ST2 and Gal3ST4 expression was comparable. Luminal mucin content correlated with sialic acid (r = 0.96; P < 0.001) or sulfate (r = 0.62; P < 0.01), but the slope of the sialic acid-derived equation was greater than that of the sulfate-derived equation, indicating a preferred increase in sialylated mucins. Third, rats were fed the control diet for 10 d while receiving antibiotic treatment. Analysis of the luminal mucin showed that sialylated mucins were more vulnerable to bacterial degradation than sulfated mucins. Finally, a study of bromo-deoxyuridine incorporation in rats fed a BF diet indicated that goblet cell proliferation accompanied by increased sialylated mucin appeared to be related to accelerated ileal epithelial cell migration. We conclude that intestinal goblet cell responses to insoluble and soluble fibers are characterized by increases in sialylated mucin production.

Uptake, transport and assimilation of 15N-nitrate and 15N-ammonium in tea (Camellia sinensis L.) plants

Abstract 15N studies were conducted using hydroponically grown tea (Camellia sinensis L.) plants to clarify the characteristics of uptake, transport and assimilation of nitrate and ammonium. From the culture solution containing 50 mg L-1 N03-N and 50 mg L-1 NH.-N, the uptake of NH3-N after 24 h was twice as high as that of NO3-N, while the uptake of N03-N from the culture solution containing 90 mg N03-N and 10 mg NH3-N was twice that of NH4-N. The presence of 0.4 mM Al had no significant effect on the N03-N and NH4-N uptake from the culture solutions containing 50 mg L-1 N03-N and 50 mg L-1 NH4-N, 90 mg L-1 N03-N and 10 mg L-1 NH4-N or 99 mg L-1 N03-N and 1 mg L-1 NH4-N. Transport of N03-derived N to young leaves was much more rapid than that of NH4-derived NO3 and NH4-derived N was largely retained in the roots and lower stem. Young and mature shoots separated from the roots absorbed more N03-N than intact plants. Nitrate assimilation occurred in both, roots and young as well as mature leaves. Internal c...

Makomotindoline from Makomotake, Zizania latifolia infected with Ustilago esculenta

Makomotindoline (1) was isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The structure was determined by the interpretation of spectroscopic data and synthesis. Makomotindoline (1), its l-Glc isomer (2) and its aglycon (3) were synthesized and their effects on rat glioma cells showed adverse effects on the cell growth.

Nitrate and oxalate contents of tea plants (Camellia sinensis L.) with special reference to types of green tea and effect of shading

Abstract To analyze the utilization of nitrate and synthesis of oxalate in tea plants Camellia sinensis L., differences in nitrate and oxalate contents in different parts (flushes, leaves, stems, and roots) and in four types of Japanese green tea (normally steamed and heavily steamed Sencha, Gyokuro, and Tencha) were examined. Nitrate content was high only in fibrous roots (0.25 g NO3 − kg−1 DW) and very low in other parts (< 0.06 g NO3 − kg−1 DW). The highest content of water-soluble oxalate was detected in flushes, whereas that of crystal oxalate (calcium oxalate) in mature leaves. Oxalate was also detected in the xylem sap. These findings suggest that oxalate, synthesized at the time of nitrate reduction in fibrous roots, is transported to flushes and leaves and then accumulates in mature leaves as crystal oxalate. There were no significant differences between the nitrate and oxalate contents of the normally steamed and heavily steamed types of Sencha, which were produced by steaming for 30 to 60 s and for 60 to 180 s, respectively. The contents of nitrate and oxalate of Gyokuro and Tencha, which were cultivated with shading, were higher than those of Sencha, which was cultivated without shading. These results suggest that shading affects the nitrate and oxalate contents of tea flushes. Nitrate and oxalate contents gradually increased in the flushes with shading but remained almost constant in the flushes without shading. At the end of the shading treatment, the nitrate content per unit field area in the flushes with shading was approximately 4-times higher than that in the flushes without shading. On the other hand, the oxalate amounts per unit field area in both flushes with and without shading increased at the same rates, indicating that the oxalate content per dry weight decreased along with growth. The ratio of oxalate content to reduced N content in the flushes under shading was higher than that in the flushes without shading. These results indicate that shading increased the nitrate content by the repression of the nitrate reductase activity and that oxalate was synthesized not only by nitrate reduction but also via other pathways.