Search

We monitored fatty acid compositions of the triacylglycerol (TAG) fractions in flax (Linum usitatissimum L.) seeds cultivated in pots with different seeding dates and nitrogen (N) fertilizing condi...

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content. Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error

We compared the protein and oil contents, seed yields, and natural abundance of 15N (σ15N) of seeds from the plants of the cultivar Enrei, which has moderate nodulating ability (Enrei) with...

The protein and oil contents in soybean seeds are major factors in seed quality. Seed proteins and oils are synthesized from sucrose and nitrogenous compounds transported into maturing seeds. In this study, we compared changes in the activity of phosphoenolpyruvate carboxylase (PEPC) and the accumulation profiles of protein and oil in maturing seeds of two soybean cultivars, which exhibit different protein and oil contents in seeds, to determine the interrelationships of them. A principal component analysis indicated a concordance of seed PEPC activity with the protein content, but did not with the oil content. PEPC activity per seed was highest in the late maturation stage, when the physiological status of the vegetative organs drastically changed. The high-protein cultivar had higher PEPC activity compared to the low-protein cultivar. These results highlight the biological role of PEPC in the synthesis of protein, therefore it was implied that PEPC could be a biomarker in soybean breeding. Abbreviations: ANOVA: analysis of variance; DS: developmental stage; DW: dry weight; FW: fresh weight; NIR: near infrared; PEP(C): phosphoenolpyruvate (carboxylase); PC(A): principal component (analysis); S.E.: standard error; WC: water content.

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.

The beneficial effects of rice bran protein (RBP), which comprises 12.8% of rice bran, have not been fully characterized. Therefore, the effects of RBP on diabetes, fatty liver, and diabetic nephropathy were investigated in Zucker Diabetic Fatty (ZDF) rats. Six-week-old male ZDF rats were fed RBP or casein (C)-based diets for 8 weeks. RBP-fed rats gained significantly more weight, but fat depot was not different from C-fed rats. Hemoglobin A1c and plasma adiponectin levels were significantly improved by consuming RBP rather than C. In RBP-fed rat livers, lipid accumulation was much lower, and the concentrations of metabolites involved in glycolysis, and betaine and carnitine metabolism were significantly higher. In addition, urinary albumin and N-acetyl-β-D-glucosaminidase excretion, and histological damage in the kidney, were significantly lower in RBP-fed rats. In conclusion, an RBP-containing diet ameliorates diabetes, fatty liver, and diabetic nephropathy in rats, compared with a C-based diet.

Raman spectroscopy was applied to characterize at the molecular scale the nutritional quality of the Japanese Koshihikari rice cultivar in comparison with other renowned rice cultivars including Carnaroli from Italy, Calrose from the USA, Jasmine rice from Thailand, and Basmati from both India and Pakistan. For comparison, two glutinous (mochigome) cultivars were also investigated. Calibrated and validated Raman analytical algorithms allowed quantitative determinations of: (i) amylopectin and amylose concentrations, (ii) fractions of aromatic amino acids, and (iii) protein content and secondary structure. The Raman assessments non-destructively linked the molecular composition of grains to key nutritional parameters and revealed a complex intertwine of chemical properties. The Koshihikari cultivar was rich in proteins (but with low statistical relevance as compared to other investigated cultivars) and aromatic amino acids. However, it also induced a clearly higher glycemic impact as compared to long-grain cultivars from Asian countries. Complementary to genomics and wet-chemistry analyses, Raman spectroscopy makes non-destructively available factual and data-driven information on rice nutritional characteristics, thus providing customers, dietitian nutritionists, and producers with a solid science-consolidated platform.

Analytical algorithms based on Raman spectroscopy are proposed for the determination of amylopectin and amylose concentrations in polished white rice, and applied to characterize and compare linear and branched polysaccharide structures in nine different types of Japanese rice. A selected algorithm used symmetric bending vibrations of the C O C glycosidic linkage from a relatively narrow spectral zone between 830 and 895 cm−1. It specifically compared the intensity of Raman signals from two types of bending common to both starch components (C1-O-C5 and C1-O-C4 at 868 and 855 cm−1, respectively) and that at the branch point peculiar to amylopectin (C1-O-C6 at 844 cm−1). Raman data were confronted with data collected by conventional amylose-iodine colorimetry method. Consistency was found between Raman and colorimetric methods over the entire series of tested rice cultivars, thus validating the newly proposed spectroscopic algorithm. The amylose content of the tested rice species broadly varied between 1.2 and 20.4%. The proposed Raman algorithm allows fast and nondestructive determination of amylose content in rice with minimal sample preparation. These characteristics might be key in the development of portable Raman devices capable to promptly screen polysaccharides in different rice cultivars with respect to their interannual and plantation-related fluctuations.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon fixation enzyme which probably plays crucial roles in seed development. A greater number of PEPC isoforms are encoded in the soybean genome, while most of the PEPC isoforms are functionally unknown. In this study, we investigated on soybean PEPC expressed in the external layer of seed coat (ELSC) during seed formation. PEPC activity in ELSC ranged from 0.24 to 1.0 U/g F.W., which could be comparable to those in whole seeds at U per dry matter. Public RNA-Seq data in separated soybean seed tissues revealed that six plant-type PEPC isogenes were substantially expressed in ELSC, and Gmppc1 and Gmppc7 were highly expressed in hourglass cells of ELSC. Gene Ontology enrichment of co-expressed genes with Gmppc1 and Gmppc7 implicated a role of these isogenes in assisting energy production and cellulose biosynthesis. Comparison of PEPC sequences from 16 leguminous species hypothesized adaptive evolution of the Gmppc1 and Gmppc7 lineage after divergence from the other plant-type PEPC lineages. Molecular diversification of these plant-type PEPC was possibly accomplished by adaptation to the functions of the soybean seed tissues. This study indicates that energy demand in immature seeds may be a driving force for the molecular evolution of PEPC.

High temperature stress during ripening increases the frequency of chalky grains, resulting in a lower market value for rice (Oryza sativa L.). Changes in starch properties and the accumulation pattern of storage proteins are proposed to be related to the occurrence of chalky grains. This study investigated changes in the accumulation of key storage compounds in the grains of Japanese highly palatable rice cultivars, subjected to high temperature stress when grown in a growth chamber and the field. The 13 kDa prolamin content was significantly reduced in a highly heat-sensitive cultivar, Tsukushiroman, whereas the 13 kDa prolamin content was not affected in a heat-tolerant cultivar, Genkitsukushi, even in a high temperature chamber condition (31/26 °C day/night), when compared with the control chamber condition (26/21 °C day/night) for both genotypes. In addition, grains grown in field conditions revealed that severely chalky grains had less 13 kDa prolamin than perfect grains in all five genotypes. Changes in amylose content and the distribution of amylopectin chain lengths did not explain the difference in grain appearance both for chamber and field experiments. These results strongly suggest that physiological processes linked with the synthesis of 13 kDa prolamin are associated with grain appearance in Japanese highly palatable under high temperature stress.

A method was developed for determination of free fatty acids (FFAs) in plasma by gas chromatography. Plasma was extracted with 3 volumes of methanol. Most cholesterol esters and triacylglycerols did not dissolve in the aqueous methanol. FFAs in the crude lipid solution were directly and selectively methylated with (trimethylsilyl)diazomethane at room temperature. Fatty acid methyl esters (FAMEs) formed were extracted with hexane, and nonreactive phospholipids were washed out with 95% methanol. The partially purified FAME preparation was analyzed by gas chromatography. The composition and amount of plasma FFAs closely approximated those obtained using two different methods.

High-quality green tea is produced from buds and young leaves grown by the covering-culture method, which employs shading treatment for tea plants (Camellia sinensis L.). Shading treatment improves the quality of tea, but shaded tea plants undergo sudden exposures to high light (HL) at the end of the treatment by shade removal. In this study, the stress response of shaded tea plants to HL illumination was examined in field condition. Chl a/b ratio was lower in shaded plants than nonshaded control, but it increased due to exposure to HL after 14 days. Rapid decline in Fv/Fm values and increases in carbonylated protein level were induced by HL illumination in the shaded leaves on the first day, and they recovered thereafter between a period of one and two weeks. These results revealed that shaded tea plants temporarily suffered from oxidative damages caused by HL exposure, but they could also recover from these damages in 2 weeks. The activities of antioxidant enzymes, total ascorbate level, and ascorbate/dehydroascorbate ratio were decreased and increased in response to low light and HL conditions, respectively, suggesting that the upregulation of antioxidant defense systems plays a role in the protection of the shaded tea plants from HL stress.

We isolated two cDNAs of phosphoenolpyruvate carboxylase (PEPC) from developing rice seeds,Osppc1andOsppc3. The deduced amino acid sequences of both cDNAs share several conserved motifs with other non-photosynthetic PEPCases, and these common motifs are known to be functionally important to their regulatory properties. The deduced protein sequence ofOsppc1was clustered into a monocotyledonous plant-specific clade, andOsppc3was clustered into a gramineous plant-specific clade in the phylogenetic tree of plant PEPCases. The mRNA accumulations ofOsppc1andOsppc3were found in developing rice seeds throughout the grain-filling stages, although their expression patterns differed:Osppc1was strongly expressed at 7 d after flowering, andOsppc3was strongly expressed at 4 d after flowering. The kinetic properties of the Osppc1 recombinant protein were quite similar to those of maize root-type PEPCase, except that the sensitivity for malate at pH 7.3 was weaker. Mining rice microarray data, we observed thatOsppc1was co-expressed with aspartate aminotransferase and alanine aminotransferase, which are involved in seed nitrogen metabolism. Moreover, reannotation of the co-expressed genes revealed thatOsppc1, the two aminotransferases and the enolase were mapped on to the consecutive reaction from 2-phosphoglycerate to glutamate and pyruvate in the cytosol. These results imply thatOsppc1functions cooperatively with the two aminotransferases in the synthesis of amino acids that are used for storage protein synthesis in developing rice seeds.

Wheat seed storage protein is of great importance for human food. To increase the contents of storage proteins effectively, nitrogen fertilizer at flowering stages is commonly applied. In our previous study, rice phosphoenolpyruvate carboxylase (PEPCase) activity in developing seeds was observed in response to nitrogen application at a flowering stage and was positively correlated to the response of the protein content in seeds of six cultivars. This observation might indicate that the seeds have a biological system for accepting nitrogen in seeds by using PEPCase. To test whether this physiological event occurs in wheat, we examined the PEPCase activity and protein content in field-grown wheat seeds under different nitrogen supply conditions. With only basal dressing, seeds showed lower PEPCase activity and protein content (both 0.90-fold) compared to seeds without basal fertilizer. With ammonium sulfate application at 8.3 and 25 g/m2 at a flowering stage, seeds showed higher PEPCase activity (1.08- and 1.17-fold, respectively) and protein content (1.15- and 1.42-fold, respectively), depending on the nitrogen level. We investigated the relationship between PEPCase activity and protein content in the seeds among four conditions. The effect of the nitrogen supply on PEPCase activity during grain-filling stages was validated by the results of a hydroponic culture experiment. Together the results demonstrate that our hypothesis seems to apply to field-grown wheat.

RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.

We investigated the regulation mechanism of cyclic electron flow around photosystem I (CEF-PSI) in the rice leaves which suffered from photosystem II (PSII) photoinhibition. High-light (HL) treatment [2000 µmol photon m–2 s–1 at 0% carbon dioxide (CO2), 2% oxygen (O2) and 25°C] of rice leaves decreased both the maximum quantum efficiency of PSII (Fv/Fm) and the light-dependent O2-evolution rate [V(O2)]. High-light treatment did not affect the relative electron flux in PSI [Φ(PSI) × PFD]. In non-treated leaves, increasing in the photon flux density (PFD) enhanced V(O2), Φ(PSI) × PFD and the ratio of oxidized P700 to total P700 [(P700+)/(P700)total]. Φ(PSI) × PFD continued to increase even after the saturation of V(O2) against PFD. These results suggested that the electrons not used for the major electron sink, photosynthetic carbon reduction-cycle, did turnover in PSI, that is, CEF-PSI functioned at a higher PFD. High-light treatments did not affect the activity of CEF-PSI and increased (P700+)/(P700)total...

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.

High-temperature stress during grain ripening facilitates the formation of chalky grains through loose packing of amyloplasts. In the present study, the changes in water distribution in the developing caryopses by high-temperature stress were investigated by magnetic resonance imaging. Milky-white and white-cored types of chalky grains, which had chalkiness around the centre of the endosperm, were formed in a high percentage in a high-temperature condition by the middle dry-matter accumulating stage. Magnetic resonance images of the early stage caryopses in the high-temperature condition showed a lower water content around the centre of the endosperm, where the disorganized development of amyloplasts was observed by scanning electron microscopy. Transcripts for α-amylase were, however, not detected there in the early and middle stage, indicating that starch degradation by α-amylase was not the cause of the formation of these types of chalky grains. In the middle stage, water content in the central chalky part became higher than in the lateral translucent part. Loose packing of amyloplasts in the chalky part may allow pooling of water in the free spaces leading to the higher water content after the middle stage. Together with the results, the physiological mechanism for the formation of white-cored and milky-white grains that occurred by high-temperature stress was discussed.

Prolamins, a group of rice (Oryza sativa )s eed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to prolamin (prolamin– GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The prolamin–GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that prolamin–GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of prolamin–GFP fusion proteins does not depend on the tissues, suggesting that the prolamin–GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

An improved method for the extraction of storage proteins from rice endosperm under conditions safe for producing food was developed. The contribution of the protein extracts to the eating quality of cooked rice and to the aroma and flavor of sake was examined. Sensory analysis was performed to evaluate the eating quality of cooked rice enriched with the protein extracts. Prolamin-enrichment increased the hardness of cooked rice, and glutelin-enrichment degraded the appearance of cooked rice. Physical analyses confirmed that prolamin-enrichment changed, whereas the glutelin-enrichment did not change the physical properties of cooked rice. Light and scanning electron microscopy of rice protein extracts revealed small particles of the prolamin extract and larger aggregated particles of the glutelin extract; these features remained after heating. The aroma and flavor of sake were negatively affected by the addition of the protein extracts. Especially, addition of prolamin significantly lowered the e...

Rice seed endosperm has two types of protein bodies (PB). Type I protein body (PB-I) accumulates prolamin and is hard to digest, while type II protein body (PB-II) mainly consists of glutelin, an easily digestible protein. A simple method to process rice protein and improve its digestibility was tested from the viewpoint of its application to food manufacturing. Rice protein prepared by alkaline extraction followed by neutralization sedimentation (AE-RP) was compared with that prepared by starch degradation by alpha-amylase (SD-RP). The crude protein content of AE-RP and SD-RP was 84.7% and 78.2%, respectively. There were no major differences in protein composition among AE-RP, SD-RP and rice flour by SDS-PAGE, except 16 kDa polypeptide. With respect to amino acids, all the groups showed quite similar compositions, although cysteine and methionine were lower in AE-RP. In an in vitro digestion study with pepsin and pancreatin, both the SDS-PAGE analysis of protein pattern and the crude protein content of undigested residue clearly demonstrated that AE-RP has a higher digestibility than SD-RP. To find the cause of the difference in digestibility, the structural property of protein bodies by two production methods was compared using electron microscopy. PB-II of AE-RP was transformed into small, amorphous granules, while that of SD-RP was still kept partial protein body structures. PB-I of AE-RP kept its protein body structure, but produced double layers. From the finding that glutelin-gold was detected by immunochemistry not only in small, amorphous granules but also in PB-I, mainly the cortex layer, in AE-RP, it became clear that PB-I was swollen and fragile as a result of alkali treatment. These results strongly indicate that the improvement in digestibility of AE-RP is a result of the structural change of PB-I and -II caused by alkaline extraction.

Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin–GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin–GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

We found novel vesicles derived from rough endoplasmic reticulum (ER) in rice endosperm. The novel vesicles had characteristic structures different from that of the ER-derived protein body type I and the Golgi-derived dense vesicles. Immunocytochemical analysis revealed that the novel vesicles are derived directly from the aggregates of vacuolar storage proteins in the rough ER. In addition, BiP, an ER-resident molecular chaperone, was localized in the novel vesicles, but also in protein storage vacuoles (PSVs). These results suggest that the novel vesicles mediate transport of vacuolar storage proteins directly from the ER to PSVs in rice endosperm.

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.

Vital protoplasts were isolated from developing dwarf rice endosperms about 10 days post anthesis. A yield of 0.98x105±0.12 protoplasts was obtained from 10 developing grains. These protoplasts were viable for at least three days in the culture medium. Transient expression of the green-fluorescent protein (GFP) was detected after four hours of introduction of the GFP gene by the polyethylene glycol (PEG)-mediated DNA transfer. As expected, the fluorescence of GFP without the targeting sequence was observed diffusely in the cytoplasm. On the other hand, the fluorescence of the fusion protein for rice prolamin followed by the GFP was observed in the small particles and the ER-network. These endosperm protoplasts had no autofluorescence upon excitation with 490 nm (blue) light. The vital protoplasts obtained from this dwarf rice variety are very useful for the physiological, biochemical and molecular biology studies of rice endosperm.

To compare the distribution of storage proteins in low-glutelin rice seed with that in other cultivars having normal protein compositions, immunofluorescence labeling with specific antibodies was applied to visualize the distribution of storage proteins in endosperm tissues. The endosperm tissues from five cultivars were reacted with anti-prolamin and anti-glutelin antibodies, and then observed by light microscopy and confocal laser scanning microscopy (CLSM). In low-glutelin rice, using microscopic analysis, a large proportion of storage proteins was observed in the endosperm tissue of 70% polished rice. To determine the localization of two types of protein bodies in endosperm tissues, images of the distribution of the type I protein body (PB-I) and the type II protein body (PB-II) were obtained by CLSM. The CLSM images showed that, in low-glutelin rice, prolamin which accumulates in PB-I remains in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. However, the other cultivars mostly contained glutelin which accumulates in PB-II and is distributed throughout the endosperm tissues. This shows that low-glutelin rice differs from the other cultivars not only in the major storage protein composition but also in the distribution of storage proteins in endosperm tissues.

Rice has storage proteins, e.g., glutelin, globulin and prolamin, in the seeds, which are used as nitrogen sources during germination. Rice prolamin has been reported to be an indigestible protein that decreases the nutritional value of rice. However, the causes for the indigestibility of prolamin are currently not clear. The objective of this study was to determine if prolamin is naturally indigestible or if cooking affects its digestibility. The gastrointestinal (GI) transit of rice 23 kDa glutelin (23G) and 13 kDa prolamin (13P) in Wistar/ST rats fed raw rice (RR) and cooked rice (CR) diets was assessed using Western blot analysis. We also measured the excretion of these proteins in the feces of these rats. Additionally, morphological observation of the structure of type-I protein bodies in the feces was performed using electron microscopy. Assessment of GI transit revealed that 23G rapidly disappeared from the GI contents of both the RR and CR groups, but 13P accumulated in the cecum of the CR group. In the CR group, prolamin, maintaining the structure of PB-I, was fully excreted in the feces. These results indicate that rice prolamin is not indigestible by nature, but is rendered indigestible by cooking.

An alcohol-soluble storage protein, a 16.6-kDa prolamin found in rice seeds, was purified from both the total protein body and purified type I protein body fractions. The partial amino acid sequences of three tryptic peptides generated from the purified polypeptide were analyzed. A part of the 16.6-kDa prolamin cDNA was amplified from developing seed mRNA by the reverse transcribed polymerase chain reaction using an oligo (dT) primer and a primer which was synthesized based on the partial amino acid sequence. The amplified product was used to isolate the full-length cDNA clone (lambda RP16) from a developing seed cDNA library. The cDNA has an open reading frame encoding a hydrophobic polypeptide of 149 amino acids. The polypeptide was rich in glutamine (20.0%), cysteine (10.0%), and methionine (6.9%). The cysteine content was higher than those of most other rice storage proteins. Messenger RNA of the 16.6-kDa prolamin was detected in seeds, but not in other aerial tissues.

The oxidative stress response of rice cytosolic ascorbate peroxidase (APX) was examined. The transcript level of cytosolic APX was significantly increased when suspension cultures of germinating rice embryos were treated with paraquat (7.9-fold) or H 2O: (6.1-fold). Induction by paraquat reached a maximum at 8 h. Induction by H2O2 peaked earlier at 4 h of treatment. This result suggests that the induction by paraquat might be caused by the H2O2 generated from superoxide. Treatment with a superoxide dismutase inhibitor, diethyldithiocarbamate, which is supposed to decrease the cellular H2O2 level, reduced the paraquat induction of cytosolic APX. In contrast, when APX and catalase were inhibited by hydroxyurea or aminotriazole, cellular H2O2 content was elevated and cytosolic APX mRNA was markedly increased without paraquat or H2O2 treatment. This suggests that cytosolic APX is regulated by the H2O2 level within cells. An increase in H 2O2 content was observed in the paraquat treated embryos, suggesting that paraquat induction of cytosolic APX was caused by H 2O2 generated through superoxide dismutation. These results indicate that H2O2 is involved in oxidative stress signalling, leading to the induction of cytosolic APX.

The 13kDa prolamins, one of the rice storage proteins, consist of complex mixtures of polypeptides encoded by multigene family that show heterogeneity both in size and solubility. Although many researchers have isolated prolamin cDNA clones, it has not been possible to correlate most of these cDNA clones with individual 13kDa prolamin mature polypeptides. We isolated three new prolamin cDNA clones, λRM1, λRM4 and λRM9. Further more, we purified six 13kDa prolamin polypeptides from rice type I protein bodies, and determined these amino acid sequences. Here we demonstrate a classification for the 13kDa prolamin polypeptides which can be divided four classes, 13-I, 13-IIa, 13-IIb and 13-III. Cysteine labeling of the prolamin polypeptides indicated that 13-I contains cysteine residues, but 13-IIa or 13-IIb have no Cysteine residues. The 13-I polypeptide was soluble in nonreducing solution when their cysteine residues form inteamolecular disulfide bonds, but not soluble at intermolecular bonding.

There are technical difficulties in obtaining intact sections of cereal grains in which mature cells and their subcellular structures are well preserved. Here we describe a simple method for sectioning hard mature rice grains. It makes possible accurate localization of storage proteins in high-quality histological sections of rice endosperm.

Summary Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.

KEY MESSAGE : We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin-GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin-GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.

The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

A cDNA library constructed from mRNAs obtained from developing rice endosperm was screened with a cDNA clone (lambda RM7) of highest frequency of occurrence (1.8%). The translation product directed by the mRNA which was hybrid-released from lambda RM7 cDNA in a wheat germ cell-free system showed a molecular size of 13 kDa when coexisting with the protein body fraction of developing maize endosperm. A polypeptide sequence composed of 156 amino acids was deduced from the nucleotide sequence. By comparison with the 19 N-terminal amino acids obtained from Edman degradation of the isolated rice 13 kDa prolamin fraction, the signal sequence was determined as consisting of 19 amino acids. The deduced polypeptide is rich in hydrophobic amino acids such as Leu and Val, and also in Gln, but lacks Lys. Hence, the amino acid composition is consistent with that of rice 13 kDa prolamin. By homology with previously reported cereal prolamins, only a single octapeptide sequence, Gln-Gln-Gln-Cys-Cys-Gln-Gln-Leu, which was observed in 15 kDa and 27 kDa zein, B- and gamma-hordein, alpha/beta- and gamma-gliadin, and gamma-secalin was conserved in the rice 10 kDa and 13 kDa prolamin. No repetitive sequences and/or sequences homologous to other cereal prolamins, except the above octapeptide, were observed for the mature 13 kDa prolamin polypeptide. The signal sequence region of the 13 kDa prolamin, however, shows homology of more than 65% in both the nucleotide sequence and the amino acid sequence with rice 10 kDa prolamin and maize zein.

A gene encoding the 13-kDa prolamin polypeptide was isolated from a rice genomic library (lambdaEMBL3) and the nucleotide sequence of an about 3-kbp EcoRI fragment was analyzed. The cloned gene (NRP33) codes for a protein composed of 156 amino acids, including a signal peptide of 19 amino acid residues and no intron is present in the genomic clone. The nucleotide sequence contains consensus TATA and CAAT boxes, and two polyadenylation signals. In addition, there are two conserved sequences named the -- 300 element and 10 consecutive repeats of the trinucleotide ATT in the 5' noncoding sequence.

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP- d -glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. AB079064 ), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP- d -glucuronic acid to UDP- d -xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Increased Stress Tolerance of Transgenic Tobacco Plants that Overexpress Ascorbate Peroxidase in Chloroplasts