Takeshi Sakaki

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress.

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are β‐glucosyl ceramides consisting of a saturated α‐hydroxy or non‐hydroxy fatty acid and a Δ4,8‐diunsaturated, C9‐methyl‐branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol‐β‐D‐glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach. Copyright

Adaptive alterations in the activities of scavengers of active oxygen in cucumber cotyledons irradiated with UV-B

Summary UV-B (290–320 nm) irradiation considerably reduced the cotyledon size of cucumber ( Cucumis sativus L.) seedlings at 20 °C, and the extent of growth inhibition was reduced at 25 °C. At both temperatures, levels of endogenous scavengers and activities of active oxygen-scavenging enzymes were affected by UV-B irradiation. In particular, ascorbate peroxidase activity increased considerably, suggesting that active oxygen species might participate in the growth inhibition induced by UV-B irradiation. However, since no positive correlation was detected between the dependence of growth inhibition on temperature and the capacity to scavenge active species of oxygen, other mechanisms must be involved in the changes in the responses to UV-B that are related to temperature.

Peroxidation of lipids and growth inhibition induced by UV-B irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumis sativus L.) were cultured in vitro under UV radiation at different wavelengths, obtained by passage of light through cut-off filters with different transmittance properties. Growth and the synthesis of chlorophyll (Chl) in cotyledons were inhibited and malondialdehyde was accumulated upon irradiation at wavelengths below 320 nm. Exogenous application of scavengers of free radicals reversed the growth inhibition induced by UV-B. Measurement of the fluorescence of Chl a suggested that electron transfer in photosystems was affected by UV-B irradiation. On the basis of these results, the involvement is postulated of active species of oxygen in damages to thylakoid membranes and the growth inhibition that are induced by UV-B irradiation.

Biosynthesis of polyunsaturated lipids in the diatom, Phaeodactylum tricornutum

The marine diatom, Phaeodactylum tricornutum, was pulse-labelled and chased with [1-14C]acetate, [1-14C] 18:1 (n-9), [1-14C] 18:2 (n-6) or [1-14C] 20:4 (n-6). Changes of radioactivities in lipid classes and molecular species were examined during the chase. The results obtained showed that phosphatidylcholine (PC) and monogalactosyidiacylglycerol (MGDG) were actively involved in the synthesis of polyunsaturated fatty acids by P. tricornutum. The changes in labelled PC during the chase suggest that 18:1 (n-9) synthesized de novo is successively desaturated to 18:4 (n-3) through the (n-3)-family in PC-linked processes, but C18 polyunsaturation was unfavourable in MGDG-linked processes. The synthesis of 20:5 (n-3) from 18: 1 (n-9) in PC suggests that 18:4 (n-3), and probably 18:3 (n-3), are released from PC to be elongated to 20:4 (n-3) and 20:3 (n-3), respectively, then desaturated to 20:5(n-3) in PC after the esterification. The synthesis of 20:5 (n-3) from 20:4 (n-6) in PC and the synthesis of 20:5 (n-3) from 18: 1 (n-9) in PC, together with the desaturation of 20:4 (n-3) to 20: 5 (n-3) in sn-2 of PC, suggests that the synthesis of 20:5 (n-3) from both 20:4 (n-3) and 20:4 (n-6) occurs in PC, although the synthesis from 20:4 (n-3) seems to occur exclusively in sn-2 of PC and that from 20:4 (n-6) in both sn-1 and sn-2 of PC. Changes of radioactivities in molecular species of PC and MGDG during the chase suggest the desaturation of 20:5 (n-3)/20:4 (n-3)-PC to 20: 5 (n-3)/20:5 (n-3)-PC, that of 20:5 (n-3)/18: 1 (n-9)-PC to 20:5 (n-3)/18:2 (n-6)-PC and successive desaturation of 20:5 (n-3)/16:1-MGDG to 20:5 (n-3)/16:3-MGDG. The results also indicated that the labelled molecular species of 20:5 (n-3)/16:1 and 20:5 (n-3)/18: 1 (n-9) occurred in both PC and MGDG, leading to the view that 20: 5 (n-3)/16: 1-PC is converted to 20:5 (n-3)/16: 1-MGDG and 20:5 (n-3)/18:1 (n-9)-PC is converted to 20:5 (n-3)/18:1 (n-9) -MGDG, as occurs in the eukaryotic pathway in higher plants. Based on the results mentioned above, we propose a tentative pathway for the synthesis of 20: 5 (n-3) from 18: 1 (n-9).

Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco

Galactolipids play an important role in plant salt tolerance through maintaining chloroplast structure and function. In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant’s response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.

Alteromonas gracilis sp. nov., a marine polysaccharide-producing bacterium.

A novel exopolysaccharide-producing bacterium, designated strain 9a2(T), was isolated from Pacific Ocean sediment. The strain was Gram-stain-negative, motile, strictly aerobic, oxidase- and catalase-positive, and required NaCl for growth. Its major isoprenoid quinone was ubiquinone-8 (Q-8), and its cellular fatty acid profile consisted mainly of C16 : 1ω7c, C18 : 1ω9c and C16 : 0. The DNA G+C content was 46.6 mol%. 16S rRNA gene sequence analysis suggested that strain 9a2(T) is a member of the genus Alteromonas . Strain 9a2(T) exhibited closest phylogenetic affinity to Alteromonas macleodii NBRC 102226(T) (99.3% 16S rRNA gene sequence similarity), A. marina SW-47(T) (99.3%), A. litorea TF-22(T) (99.0%), A. australica H17(T) (98.7%), A. simiduii BCRC 17572(T) (98.5%), A. stellipolaris LMG 21861(T) (98.3%) and A. hispanica F-32(T) (98.2%). The DNA-DNA reassociation values between strain 9a2(T) and A. macleodii JCM 20772(T), A. marina JCM 11804(T), A. litorea JCM 12188(T), A. australica CIP 109921(T), A. simiduii JCM 13896(T), A. stellipolaris LMG 21861(T) and A. hispanica LMG 22958(T) were below 70%. Strain 9a2(T) contained phosphatidylethanolamine, phosphatidylglycerol and an unidentified polar lipid. Owing to differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequences and DNA-DNA relatedness data, the isolate merits classification as representing a novel species, for which the name Alteromonas gracilis sp. nov. is proposed. The type strain of this species is 9a2(T) ( =JCM 30236(T) =NCIMB 14947(T)).

Lipids and fatty acids in guard-cell protoplasts from Vicia faba leaves

Guard-cell and mesophyll-cell protoplasts were isolated from Vicia faba leaves, and their lipids and fatty acids quantitatively analysed. All the glycerolipids and fatty acids that occurred in mesophyll-cell protoplasts were found in guard-cell protoplasts. On a total fatty acid basis, levels of chloroplast lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol) and phosphatidylglycerol were lower and their constituent fatty acids were more saturated in guard-cell protoplasts than in mesophyll-cell ones. In contrast, levels of extrachloroplastic lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) were higher in guard-cell protoplasts than in mesophyll-cell protoplasts. These results suggest that extrachloroplastic membrane systems, including mitochondria, are more developed than chloroplast membranes. In addition, guard-cell protoplasts contained a large amount of triacylglycerols, a probable component of spherosomes, lipid droplets and/or plastoglobuli, which are frequently observed in guard cells.

Psychrobacter oceani sp. nov., isolated from marine sediment

A novel marine bacterium, designated strain 4k5(T), was isolated from a sediment sample of the Pacific Ocean. The strain was Gram-stain-negative, strictly aerobic, non-motile, oxidase-positive and catalase-positive and required Na(+) for growth. Its major isoprenoid quinone was ubiquinone 8 (Q-8), and its cellular fatty acid profile consisted mainly of C18 : 1v9c (71.4%), C16 : 1v7c (9.1%) and C18 : 0. The DNA G+C content was 45.3 mol%. 16S rRNA gene sequence analysis suggested that strain 4k5(T) is a member of the genus Psychrobacter . Strain 4k5(T) exhibited the closely phylogenetic affinity to Psychrobacter pacificensis IFO 16270(T) (99.4% 16S rRNA gene sequence similarity), P. piscatorii T-3-2(T) (97.7%), P. nivimaris 88/2-7(T) (97.7%), P. celer SW-238(T) (97.7%), P. aestuarii SC35(T) (97.6%) and P. vallis CMS39(T) (97.6%). DNA-DNA hybridization between strain 4k5(T) and P. pacificensis NBRC 103191(T), P. piscatorii JCM 15603(T). P. nivimaris DSM 16093(T), P. celer JCM 12601(T), P. aestuarii JCM 16343(T) and P. vallis DSM 15337(T) was 42.5, 47.0, 38.1, 23.7, 9.0 and 27.4%, respectively. Owing to the significant differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA relatedness data, the isolate merits classification within a novel species, for which the name Psychrobacter oceani sp. nov. is proposed. The type strain is 4k5(T) ( = JCM 30235(T) =NCIMB 14948(T)).

Effects of Air Pollutants on Lipid Metabolism in Plants

Ozone (O3), nitrogen oxides (NOx), peroxyacetyl nitrate (PAN), and sulfur dioxide (SO2) are major global air pollutants, causing serious vegetative damage and forest decline. To clarify both the acute and chronic phytotoxic mechanisms of these pollutants, their physiological and biochemical effects on plants have been extensively studied during the past several decades. According to ultrastructural observations of plant cells injured by these pollutants, cellular membrane systems are affected by the pollutants (Thomson 1975; Huttunen and Soikkeli 1984), and membrane permeability is also seen to change after treatment with SO2 (Malhotra and Hocking 1976) and O3 (Heath and Castillo 1988). It is now generally accepted that cellular membranes are among the primary sites of pollutant attack and, since lipids are important membrane components and play essential roles in maintaining membrane structure and function, many workers have examined the effects of pollutants on lipids to clarify the mechanisms of their phytotoxicity (Mudd et al. 1984; Heath 1984; Sakaki 1998).