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HAK family transporters primarily function as K+ transporters and play major roles in K+ uptake and translocation in plants, whereas several HAK transporters exhibit Na+ transport activity. OsHAK2, a rice HAK transporter, was shown to mediate Na+ transport in Escherichia coli in a previous study. In this study, we investigated whether OsHAK2 is involved in Na+ transport in the rice plant. Overexpression of OsHAK2 increased Na+ translocation from the roots to the shoots of transgenic rice. It also increased both root and whole-plant Na+ content, and enhanced shoot length under low Na+ and K+ conditions. Meanwhile, OsHAK2 overexpression increased salt sensitivity under a long-term salt stress condition, indicating that OsHAK2 is not involved in salt tolerance, unlike in the case of ZmHAK4 in maize. These results suggest that OsHAK2 is permeable to Na+ and contributes to shoot growth in rice plants under low Na+ and K+ conditions.

Nickel (Ni) is an essential micronutrient for plants although it is considered toxic when present in excess in the soil. This study investigated the transfer of Ni from the soil to rice in terraced paddy fields affected by serpentinite, which contains an anomalously higher Ni content compared with other geological materials. Soils, soil solutions, and rice plants were collected at several different growing stages from three adjacent terraced paddy fields subject to the same water and fertilizer management. Temporal changes in their elemental compositions revealed that a higher concentration of Ni was dissolved in the soil solution during flooded conditions, probably due to the co-solubilization with Mn oxides under low redox potential conditions. However, rice accumulated Ni at a higher rate during the drainage period than in the flooding period. Although the Ni concentration in the soil solution was lowest in the drainage period, the relative concentration to Fe (i.e., Ni/Fe ratio) was much higher than that in flooded conditions. These relationships suggest that a potential measure to counter the transfer of Ni from the soil to rice in serpentine-affected paddy fields is to increase Fe phytoavailability during the drainage period.

Shade cultivation of tea plants (Camellia sinensis L.) is employed for the production of high-quality green tea which increases the content of chlorophylls and free amino acids, including theanine. However, shaded tea plants suffer from photooxidative stress caused by sudden exposure to high light (HL) when the shade is removed. In this study, we tried to acclimatize shaded tea plants to light prior to shade removal to alleviate HL-induced stress. Acclimated tea plants showed milder photoinhibition in response to HL exposure than the shaded plants without acclimation. Moreover, there were no large differences in the total chlorophylls and free amino acids (including theanine) content between acclimated and non-acclimated plants. These results indicate that acclimation of shaded tea plants can alleviate subsequent HL stress without causing large changes in the content of chemical components associated with tea quality.

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.

Key message Laser microdissection applied on the developing rice endosperm revealed tissue- and stage-specific regulators modulating programmed cell death and desiccation tolerance mechanisms in the central starchy endosperm following starch metabolism. Abstract Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44 K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

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.

Phospho

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.

Rice prolamin species form a layered structure in the protein body type I (PB-I) storage organelle. Rice prolamins are classified as 10 kDa, 13a-1, 13a-2, 13b-1, 13b-2 and 16 kDa prolamin. Prolamin species form layer structure in PB-I in order of 10 kDa core, 13b-1 layer, 13a (13a-1 and 13a-2) and 16 kDa middle layer and 13b-2 outer-most layer. In a previous study, we showed that the fusion proteins in 13b-2 prolamin-GFP, 13a-1 prolamin-GFP and 10 kDa prolamin-GFP were localized in the same layer of PB-I as the native prolamin, when they were expressed by their respective native prolamin promoters. Our preliminary study suggested that the temporal control of the native prolamin promoters was responsible for the localization of the respective prolamins. The aim of this study was to determine whether the use of a prolamin promoter other than the native prolamin promoter would change the localization of prolamin-GFP fusion proteins. For this purpose, we generated transgenic lines expressing 13b-2 prolamin-GFP and 13a-1 prolamin-GFP fusion proteins driven by each prolamin promoter other than the native prolamin promoter. As a result, the localization of the fusion protein in PB-I was changed. Based on our results, foreign protein localization in PB-I can be achieved by the temporal control of the different prolamin promoters.

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.

Heat stress occurrence during seed filling leads to the formation of a chalky portion in the limited zone of the starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of a 44 K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. The most distinct expression pattern was observed in modules where most of the small heat shock proteins and cellular organization genes were changed under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones. The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central starchy endosperm zone, preferential down-regulation of high molecular weight heat shock proteins (HMW HSPs), including a prominent member encoding endoplasmic reticulum (ER) chaperones, by heat stress was observed, while changes in expression of starch biosynthesis genes were minimal. Characterization of transgenic plants suppressing endosperm lumenal binding protein gene (BiP1), an ER chaperone preferentially down-regulated at the MW zone under heat stress, showed evidence of forming the chalky grains without disturbing the expression of starch biosynthesis genes. The present LM-based comprehensive expression analysis provides novel inferences that HMW HSPs play an important role in controlling redox, nitrogen and amino acid metabolism in endosperm leading to the formation of MW and WB chalky grains under heat 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.

Introns have an enhancing effect on gene expression known as intron-mediated enhancement in various organisms, including plants. Although the mechanism of the enhancement is largely unknown, most enhancing introns are first introns. In this study, we examined whether the first intron of rice superoxide dismutase sodCc2 gene has an enhancing effect in rice and other plant species. A transient expression assay revealed that the sodCc2 intron elevated reporter gene expression in rice, wheat, and maize, but not in Arabidopsis and tobacco, indicating that the sodCc2 intron has an enhancing effect in monocot but not in dicot plants. To find the putative signal sequences responsible for the enhancement, we carried out an in silico search and found two motifs conserved among the sodCc2 intron and 11 enhancing introns previously known in rice. The motifs contain a consensus sequence, GATCTG, which also exists in the conserved motif found in Arabidopsis enhancing introns. Our results suggest that common motifs are conserved between rice and Arabidopsis enhancing introns.

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.

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.

Carnation (Dianthus caryophyllus) flowers exhibit climacteric ethylene production followed by petal wilting, a senescence symptom. DcACS1, which encodes 1-aminocyclopropane-1-carboxylate synthase (ACS), is a gene involved in this phenomenon. We determined the genomic DNA structure of DcACS1 by genomic PCR. In the genome of 'Light Pink Barbara', we found two distinct nucleotide sequences: one corresponding to the gene previously shown as DcACS1, designated here as DcACS1a, and the other novel one designated as DcACS1b. It was revealed that both DcACS1a and DcACS1b have five exons and four introns. These two genes had almost identical nucleotide sequences in exons, but not in some introns and 3'-UTR. Analysis of transcript accumulation revealed that DcACS1b is expressed in senescing petals as well as DcACS1a. Genomic PCR analysis of 32 carnation cultivars showed that most cultivars have only DcACS1a and some have both DcACS1a and DcACS1b. Moreover, we found two DcACS1 orthologous genes with different nucleotide sequences from D. superbus var. longicalycinus, and designated them as DsuACS1a and DsuACS1b. Petals of D. superbus var. longicalycinus produced ethylene in response to exogenous ethylene, accompanying accumulation of DsuACS1 transcripts. These data suggest that climacteric ethylene production in flowers was genetically established before the cultivation of carnation.

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.

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro . Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism.

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...

The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.

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.

Interferon-a (IFN-a) is a principle cytokine that plays a key regulatory role in mammalian immune systems. The recombinant DNA of IFN-a is composed of the signal sequence region for rice 10 kDa prolamin cDNA, the amino- terminal region of b-glucuronidase DNA, and the mature polypeptide region of human IFN-aDNA, and is expected to produce a biologically active form of IFN-a. This chimerical DNA for coding IFN-a under the control of a cauliflower mosaic virus 35S promoter and a 5� -intron of rice cytosolic superoxide dismutase gene (sodcC1), was transformed into dwarf rice by an Agrobacterium-mediated system. Three lines of transgenic rice plant expressing various levels of IFN-a polypeptide were finally generated. The expression level of the recombinant polypeptide in each line was analyzed by an IFN-a antiviral activity assay and enzyme immunoassay. Higher expression of IFN-a was achieved in developing seed endosperm in two of transgenic rice lines. The replacement of the native signal peptide of IFN-a with the prolamin signal peptide was effective for transporting the IFN-a polypeptide into the ER-derived protein body of developing seed endosperm. These results suggest that rice can be used to produce many biologically active mammalian proteins that accumulate in target organelles such as protein bodies.