Nitrogen Fixation in Soybean Nodules Affects Seed Protein and Oil Contents: The Suggested Mechanism from the Coordinated Changes of Seed Chemical Compositions and Phosphoenolpyruvate Carboxylase Activity Caused by Different Types of Nitrogen Fertilizer

The contents of seed storage compounds, protein and oil, determine the best use of soybean seeds, namely materials for food processing and oil production. Genetic and environmental factors could affect the chemical compositions of soybean seeds. However, the mechanisms of how the accumulation of these primary seed compounds is regulated are mostly unclear. In this chapter, we describe the different effects of nodulation on the protein and oil contents in soybean seeds and the crucial role of phosphoenolpyruvate carboxylase (PEPC) in the protein accumulation of soybean seeds. Based on our previous studies on soybean seeds, we introduce five manners deduced; (1) protein accumulation is independent of oil accumulation, (2) nitrogen fixation results in decreasing oil amount per seed and decreased seed oil content, (3) a high pseudo negative correlation between protein and oil contents in seeds is likely to be observed under less nitrogen supply from the soil, (4) nitrogen absorbed from soil during the late growth stage promote seed production, (5) plant-type PEPC, ex. Gmppc2 in soybean could play a role in amino acid biosynthesis for storage protein accumulation in seeds during the late maturation period

Increased tolerance to photooxidative stress by overexpression of mitochondrial superoxide dismutase in transgenic tobacco

76-80Under abiotic stress conditions, plants suffer from oxidative damage caused by increased generation of reactive oxygen species (ROS). Although chloroplasts are the main source of ROS generation in plant cells, changes in ROS level in mitochondria affect plant stress response. In this study, we investigated whether overexpression of mitochondrial manganese superoxide dismutase (Mn-SOD) can enhance tolerance to photooxidative stress. We produced transgenic tobacco plants overexpressing rice Mn-SOD under the control of cauliflower mosaic virus 35S promoter, and assessed the stress tolerance of those plants. The transgenic lines showed higher total SOD activity by 2.8 to 5.2 fold than wild type plants. Chlorophyll fluorescence measurement revealed that the stress-induced inhibition of photosystem II was alleviated under high light, chilling and heat stresses in the transgenic lines. This result indicates that the Mn-SOD contributes to the defense against photooxidative damages in chloroplasts under these stress conditions

Raman Molecular Fingerprints of Rice Nutritional Quality and the Concept of Raman Barcode

The nutritional quality of rice is contingent on a wide spectrum of biochemical characteristics, which essentially depend on rice genome, but are also greatly affected by growing/environmental conditions and aging during storage. The genetic basis and related identification of genes have widely been studied and rationally linked to accumulation of micronutrients in grains. However, genetic classifications cannot catch quality fluctuations arising from interannual, environmental, and storage conditions. Here, we propose a quantitative spectroscopic approach to analyze rice nutritional quality based on Raman spectroscopy, and disclose analytical algorithms for the determination of: (i) amylopectin and amylose concentrations, (ii) aromatic amino acids, (iii) protein content and structure, and (iv) chemical residues. The proposed Raman algorithms directly link to the molecular composition of grains and allow fast/non-destructive determination of key nutritional parameters with minimal sample preparation. Building upon spectroscopic information at the molecular level, we newly propose to represent the nutritional quality of labeled rice products with a barcode specially tailored on the Raman spectrum. The Raman barcode, which can be stored in databases promptly consultable with barcode scanners, could be linked to diet applications (apps) to enable a rapid, factual, and unequivocal product identification based on direct molecular screening

電気的形質転換法を用いた cDNA ライブラリーの作製(農芸化学部門)

電気的形質転換法を用いプラスミドDNAを導入することにより大腸菌を形質転換した。我々は高効率を得る工夫をし, この方法を用いることでコンピテントセルを用いる形質転換法(Hanahan法)では出せない高効率を得ることが可能になった。ここではこの方法で登熟期イネ胚乳及びホウレンソウ芽生えよりcDNAライブラリーの作製に成功した。To transform Escherichia coli a plasmid DNA was introduced by electroporation. We improved transformation efficiency adding several chemicals, biological materials and/or preheating of the buffer solution which was added just affer electrodischarge. The improved method gave higher transformation efficiency comparing with that of Hanahan\u27s. cDNA libraries for mRNAs appearing in the developing rice seeds and spinach seedlings were constructed using this method

アフリカツメガエル卵母細胞を用いるタンパク質生合成のための簡易微量注入法(農芸化学部門)

アフリカツメガエル卵母細胞へのRNA, DNA等の細胞成分注入用の簡易な微量注入装置を考案, 試作した。この装置を用い特に植物性mRNAをアフリカツメガエル卵母細胞へ注入しタンパク質生合成を行わせた。その結果, tobacco mosaic virus RNA, brome mosaic virus RNA, イネglutelin前駆体mRNA, エンドウriburose 1,5 bis-phosphate carboxylase mRNAが忠実に翻訳され, 対応するpolypeptideが形成されることをsodium dodecyl sulfate polyacrylamide gel電気泳動(SDS-PAGE)による分子量の測定と免疫学的方法を組合わせることにより確認した。A simplified and convenient microinjection system to inject cell components such as DNA and RNA into Xenopus oocytes was devised and constructed. By use of this system, RNAs of plants were injected into Xenopus oocytes, then their protein syntheses were examined. Tobacco mosaic virus RNA, brome mosaic virus RNA, rice glutelin precusor mRNA, and pea Riburose 1,5bisphosphate carboxylase mRNA were faithfully translated. The nature of the synthesized polypeptide was confirmed by fluorography using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelctrophoresis

電気的ブロッティング法を用いる cDNA クローンの抗体スクリーニング(農芸化学部門)

大腸菌発現ベクターpUC 9を用いて作製したcDNAライブラリーから電気的ブロッティング法を用いる抗体スクリーニングを行った。ここに示す電気的ブロッティング法を使用すると, 従来の抗体スクリーニング法で障害となる非特異的吸着によるバックグラウンドをほとんど除去出来た。ここでは, 登熟期イネ種子について作製したcDNAライブラリーより, プロラミン抗体を用いてプロラミンcDNAの一つをスクリーニングする過程を例として示し, この方法の特徴を解説する。A cDNA library constructed using plasmid expression vector, pUC 9,was screened immunologically after the products of the transformed cells were electrically transfered to nitrocellulose filter. Electroblotting technic mentioned here reduced remarkably the background which interfered strongly the identification of the target clones. In this paper, an example is shown in which a clone carring rice prolamin cDNA sequence was screened from a cDNA library for developing rice seed using the electroblotting technic

A Novel cis-Element That Is Responsive to Oxidative Stress Regulates Three Antioxidant Defense Genes in Rice

All organisms have defense systems against oxidative stress that include multiple genes of antioxidant defense. These genes are induced by reactive oxygen species under condition of oxidative stress. In this study, we found that a 28-bp motif is conserved on the promoter regions of three antioxidant defense genes in rice (Oryza sativa): cytosolic superoxide dismutase (sodCc1), cytosolic thioredoxin (trxh), and glutaredoxin (grx). We demonstrated that the 28-bp sequence acts as a cis-element responsive to oxidative stress by transient expression assay and designated it as CORE (coordinate regulatory element for antioxidant defense). The CORE was activated by methyl viologen treatment and induced a 3.1-fold increase in expression of the reporter gene, but it did not respond to hydrogen peroxide. The expressions of the sodCc1, trxh, and grx genes were coordinately induced by methyl viologen, suggesting that multiple genes involved in antioxidant defense are controlled by a common regulatory mechanism via CORE. Application of the mitogen-activated protein kinase kinase inhibitor caused the constitutive induction of the sodCc1, trxh, and grx genes and the activation of CORE without methyl viologen treatment. These results indicate that a mitogen-activated protein kinase cascade is involved in the gene regulation mediated by CORE

Analysis of Storage Protein Distribution in Rice Grain of Seed-Protein Mutant Cultivars by Immunofluorescence Microscopy

Localization of storage proteins in rice grains of seed-protein mutant cultivars, low-glutelin cultivars LGC-1, LGCsoft and a 26-kDa-globulin-deficient low-glutelin cultivar LGC-Katsu, was examined by immunofluorescence microscopy using fluorescence-labeled antibodies of 13 kDa prolamin and 23 kDa glutelin. Abundant 13 kDa prolamin and 23 kDa glutelin was observed in the outer regions of rice grains. Image analysis revealed that peaks of fluorescence intensity of both proteins were located at 2–7% of the width or longitudinal length of brown rice distant from the outer surface of brown rice (RDOS) on the dorsal, ventral, and apical sides of brown rice. In these seed-protein mutant cultivars and a normal-type cultivar Nihonmasari, the foot of the distribution peaks of both proteins were located at 20–30% RDOS on the ventral and apical side. In contrast, on the dorsal side of rice grain, varietal differences of RDOS at the foot of the distribution peak of both proteins varied with the cultivar. The RDOS was 20 –40%; and Nihonmasari >LGC-1≒LGCsoft >LGC-Katsu. Although the quantity of 13 kDa prolamin and 23 kDa glutelin greatly varied with the cultivar in the outer region of rice grain, both proteins were distributed uniformly at low levels in the inner region in all cultivars. Furthermore, SDS-PAGE analysis showed that a larger quantity of 13 kDa prolamin localized on the ventral than the dorsal side of rice grains in seed-protein mutant cultivars, especially in LGC-Katsu