Phytochrome-mediated growth inhibition of seminal roots in rice seedlings

In rice ( Oryza sativa ) seedlings, continuous white-light irradiation inhibited the growth of seminal roots but promoted the growth of crown roots. In this study, we examined the mechanisms of photoinhibition of seminal root growth. Photoinhibition occurred in the absence of nitrogen but increased with increasing nitrogen concentrations. In the presence of nitrogen, photoinhibition was correlated with coiling of the root tips. The seminal roots were most photosensitive 48–72 h after germination during the 7-day period after germination. White-light irradiation for at least 6 h was required for photoinhibition, and the Bunsen–Roscoe law of reciprocity was not observed. Experiments with phytochrome mutants showed that far-red light was perceived exclusively by phyA, red light was perceived by both phyA and phyB, and phyC had little or no role in growth inhibition or coiling of the seminal roots. These results also suggest that other blue-light photoreceptors are involved in growth inhibition of the seminal roots. Fluence-response curve analyses showed that phyA and phyB control very low-fluence response and low-fluence response, respectively, in the seminal roots. This was essentially the same as the growth inhibition previously observed at the late stage of coleoptile development (80 h after germination). The photoperceptive site for the root growth inhibition appeared to be the roots themselves. All three phytochrome species of rice were detected immunochemically in roots.     

Ethylene and salicylic acid control glutathione biosynthesis in ozone‐exposed Arabidopsis thaliana

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone‐induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild‐type, Col‐0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col‐0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real‐time polymerase chain reaction using genes involved in GSH metabolism. Induction of γ‐glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col‐0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col‐0. Moreover, ozone‐induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone‐induced leaf injury by increasing de novo biosynthesis of GSH.     

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T₁ soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.     

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.     

Expression of exogenous fluorescent proteins in early freshwater pond snail embryos

We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2–3 h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8–9 h and was stable up to 24 h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.     

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.     

2-Cyclohexylcarbonylbenzimidazoles as potent,orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonists

The synthesis and biological evaluation of new potent opioid receptor-like 1 (ORL1) antagonists are presented. Conversion of the thioether linkage of the prototype [It is reported prior to this communication as a consecutive series.: Kobayashi, K.; Kato, T.; Yamamoto, I.; Shimizu, A.; Mizutani, S.; Asai, M.; Kawamoto, H.; Ito, S.; Yoshizumi, T.; Hirayama, M.; Ozaki, S.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett., in press] to the carbonyl linker effectively reduces susceptibility to P-glycoprotein (P-gp) efflux. This finding led to the identification of 2-cyclohexylcarbonylbenzimizole analogue 7c, which exhibited potent ORL1 activity, excellent selectivity over other receptors and ion channels, and poor susceptibility to P-gp. Compound 7c also showed satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Furthermore, 7c showed good in vivo antagonism. Hence, 7c was selected as a clinical candidate for a brain-penetrable ORL1 antagonist.     

Establishment of Agrobacterium tumefaciens-Mediated Transformation of an Oleaginous Fungus,Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5⁻ strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10⁸ spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.Two decades ago, a filamentous zygomycete fungus, Mortierella alpina 1S-4, was isolated from soil as a potent producer of polyunsaturated fatty acids (PUFAs) in our laboratory and was utilized for commercial production of arachidonic acid (AA) (20:4n-6) (21). Breeding of mutants derived from the wild strain led to the production of dihomo-γ-linolenic acid (20:3n-6) and Mead acid (20:3n-9) (10-12) (Fig. ​(Fig.1).1). Furthermore, we attempted to produce other PUFAs synthesized in M. alpina 1S-4, since some fatty acids (e.g., 18:2n-9, 18:4n-3, and 20:4n-3) have limited natural sources and could have promising beneficial physiological effects (9). In particular, for microbial production of n-3 PUFAs, currently prepared from fish oil, it is necessary to achieve stable productivity and quality; however, mutation treatment caused low activity of the specific enzymes involved in PUFA biosynthesis, which is unsuitable for industrial application. In addition, gene manipulation tools have not been sufficiently developed for metabolic control of the PUFA synthetic pathway. Genetic manipulation is a new means of molecularly breeding industrial strains, analyzing their physiological properties, and clarifying the biosynthetic pathway to PUFAs. A comprehensive transformation system for this fungus has been fundamentally established. It involves a uracil-auxotrophic mutant (ura5⁻ strain) as a host strain, a homologous ura5 gene as a selectable marker gene, and transformation through the biolistic method, which is the only effective method (24).Open in a separate windowFIG. 1.Putative biosynthetic pathway of PUFAs in Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; ALA, α-linolenic acid; GLA, γ-linolenic acid; SDA, stearidonic acid; EDA, n-9 eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; ETA, n-3 eicosatetraenoic acid; MA, Mead acid. Open and black arrows indicate elongase and desaturase reactions, respectively.Agrobacterium tumefaciens-mediated transformation (ATMT) has been employed for a wide range of plants (7, 27). Recently, it was reported that A. tumefaciens is also able to transfer its DNA to various fungi, including ascomycetes, basidiomycetes, zygomycetes, and oomycetes, as well as to plants (2, 5, 16). Additionally, this bacterium can transform intact cells and spores as well as protoplasts. Under mild conditions, the ATMT system generates a large number of stable transformants, which show vigorous growth, indicating that the ATMT system can be an efficient tool for molecular manipulation of M. alpina 1S-4. Moreover, the frequency of homologous recombination was higher than that with conventional transformation methods (8). In this study, we evaluated the external gene transfer system using the ATMT system and determined the optimal conditions for M. alpina 1S-4. Furthermore, we overexpressed the ω3-desaturase gene to improve n-3 PUFA productivity in an industrial n-6-PUFA-producing strain, M. alpina 1S-4 (18, 20), using ATMT.