Molecular mapping of the ge s gene controlling the super-giant embryo character in rice (Oryza sativa L.)

The giant-embryo character is useful for quality improvement in rice. Three alleles controlling embryo size have been reported at the ge locus. Based on trisomic analysis, this locus is known to reside on chromosome 7. The objective of the present study was to identify linkage between molecular markers and the ge ^s gene using an existing molecular map of rice and an F₂ population derived from Hwacheongbyeo-ge ^s (super-giant embryo)/Milyang 23. The bulked-segregant method was used to screen 38 RFLPs and two microsatellite markers from rice chromosome 7. RZ395 and CDO497 flanked the ge ^s gene, at 2.4 cM and 3.4 cM, respectively. The two microsatellite markers, RM18 and RM10, were linked with ge ^s at 7.7 cM and 9.6 cM, respectively. The availability of molecular markers will facilitate selection of this grain character in a breeding program and provide the foundation for map-based gene isolation.     

Reflectance Variation within the In-Chlorophyll Centre Waveband for Robust Retrieval of Leaf Chlorophyll Content

The in-chlorophyll centre waveband (ICCW) (640–680 nm) is the specific chlorophyll (Chl) absorption band, but the reflectance in this band has not been used as an optimal index for non-destructive determination of plant Chl content in recent decades. This study develops a new spectral index based solely on the ICCW for robust retrieval of leaf Chl content for the first time. A glasshouse experiment for solution-culture of one chlorophyll-deficient rice mutant and six wild types of rice genotypes was conducted, and the leaf reflectance (400–900 nm) was measured with a high spectral resolution (1 nm) spectrophotometer and the contents of chlorophyll a (Chla), chlorophyll b (Chlb) and chlorophyll a+b (Chlt) of the rice leaves were determined. It was found that the reflectance curves from 640 nm to 674 nm and from 675 nm to 680 nm of the low-chlorophyll mutant leaf were drastically steeper than that of the wild types in the ICCW. The new index based on the reflectance variation within ICCW, the difference of the first derivative sum within the ICCW (DFDS_ICCW), was highly sensitive (r = −0.77, n = 93, P<0.01) to Chlt while the mean reflectance (R_ICCW) in the ICCW became insensitive (r = −0.12, n = 93, P>0.05) to Chlt when the leaf Chlt was higher than 200 mg/m². The best equations of R-ICCW and DFDS_ICCW yielded an RMSE of 78.7, 32.9 and 107.3 mg/m², and an RMSE of 37.4, 16.0 and 45.3 mg/m⁻², respectively, for predicting Chla, Chlb and Chlt. The new index could rank in the top 10 for prediction of Chla and Chlt as compared with the 55 existing indices. Additionally, most of the 55 existing Chl-related VIs performed robustly or strongly in simultaneous prediction of leaf Chla, Chlb and Chlt.     

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice

Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss‐of‐function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild‐type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss‐of‐function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo–endosperm size control, the genetic network of the related genes should be elucidated.     

不同类型特种稻种质营养及功能性成分含量的差异

本研究以具有有色种皮、巨胚、甜味、香味、糯性等单一特殊性状或2个以上特殊性状聚合于一体的新创制特种稻种质39份和1份白米种质(对照)为试验材料,进行了黑米、黑褐米、红米、香糯米、黑巨胚糯米、红巨胚糯米、巨胚糯米、黑甜米、红甜米、白甜米等不同类型特种稻种质的营养及功能性成分含量的差异评价。结果表明,供试特种稻类型在大部分氨基酸含量和矿质元素含量上与白米差异不显著,只在个别氨基酸和矿质元素含量上与白米呈显著或极显著差异。黑褐米、红米、黑巨胚糯米、红巨胚糯米、巨胚糯米、红甜米和白甜米等7种类型的赖氨酸含量显著或极显著高于白米,高2.91%~24.68%;黑米、黑褐米、红米、香糯米、黑巨胚糯米和红甜米等6种类型的铁含量显著或极显著高于白米,高17.62%~68.09%;黑褐米、红米、黑巨胚糯米、红巨胚糯米、黑甜米、红甜米、白甜米等7种类型的钙含量显著或极显著高于白米,高23.56%~49.46%;黑米、黑褐米、红米、黑甜米、红甜米、白甜米等6种类型的锌含量显著或极显著高于白米,高12.21%~55.87%。由此表明,具有有色种皮、巨胚、甜味、香味、糯性等单一特殊性状或2~3个特殊性状的聚合对赖氨酸含量与铁、钙和锌含量的提高方面具有一定的增加效应,认为在今后以赖氨酸、铁、钙和锌含量为目标性状的功能性水稻育种中,多个特殊性状的聚合将是增加上述功能性成分含量的有效途径之一。通过鉴定评价,从创新种质中还筛选出一些功能性成分含量相对较高的优异种质,白甜米1553和红巨胚糯米1476的赖氨酸含量较高,比白米分别高29.37%和23.42%;红米1439和红米1440的铁含量较高,比白米分别高99.05%和80.00%;黑甜米1511和黑甜米1515的硒含量较高,比白米分别高194.14%和136.48%;白甜米1551和香糯米1446的γ-氨基丁酸含量较高,比白米分别高14.56%和11.83%;黑巨胚糯米1464和黑米1432的花色苷含量较高,比供试18份有色稻米的平均值分别高253.23%和248.83%。这些新创制的功能性成分含量较高的水稻种质有待于今后在育种、生态适应性鉴定与产业化中进一步得到利用。     

Comparison of gamma-aminobutyric acid production in Thai rice grains

Gamma-aminobutyric acid (GABA) has many pharmacological functions including being a major inhibitory neurotransmitter. Two comparative methods for GABA production in rice grains as main food source in Thailand were investigated in this study. Fermentation and germination method were separately carried out using seven selected local grain cultivars in northern Thailand. Red yeast rice, obtained from the fermentation method, gave the higher GABA concentration than the germinated rice produced from the germination method in most rice cultivars. The highest GABA concentration was 28.37 mg/g at 3 weeks fermentation time of glutinous rice, O. sativa L. cv. Sanpatong 1 cultivars, while germinated rice from glutinous rice; O. sativa L. cv. Korkor6 (RD6) cultivars contained the highest GABA concentration of 3.86 mg/g. These results provide information for the basis of an appropriate method for GABA production. The fermentation produced higher GABA concentration but required longer production period and red yeast rice was obtained as product. On the other hand, the germination method yielded rice grains with lower GABA but in more suitable form for consumption. Both methods are considered to be economical and efficient methods to increase GABA in rice grains, providing alternative products with higher nutritional values.     

Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

Background and Aims

Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings.

Methods

Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared.

Key results

In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations.

Conclusions

These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.     

Cloning,characterization and expression of \emph{OsFMO}_{{\mathbf{(}\emph{t}\mathbf{)}}} in rice encoding a flavin monooxygenase

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO _(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO _(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a ‘G×G××G’ characteristic structure. OsFMO_(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO_(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO _(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO_(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO _(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO _(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.     

Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

Seed-specific expression of truncated OsGAD2 produces GABA-enriched rice grains that influence a decrease in blood pressure in spontaneously hypertensive rats

Gamma-aminobutyric acid (GABA) is a four-carbon amino acid that is commonly present in living organisms and functions as a major inhibitory neurotransmitter in mammals. It is understood to have a potentially anti-hypertensive effect in mammals. GABA is synthesized from glutamate by glutamate decarboxylase (GAD). In plants, GAD is regulated via its calmodulin-binding domain (CaMBD) by Ca²⁺/CaM. We have previously reported that a C-terminal truncated version of one of the five rice GAD isoforms, GAD2ΔC, revealed higher enzymatic activity in vitro and that its over-expression resulted in exceptionally high GABA accumulation (Akama and Takaiwa, J Exp Bot 58:2699–2607, 2007). In this study, GAD2ΔC, under the control of the rice glutelin promoter (GluB-1), was introduced into rice cells via Agrobacterium-mediated transformation to produce transgenic rice lines. Analysis of the free amino acid content of rice grains revealed up to about a 30-fold higher level of GABA than in non-transformed rice grains. There were also very high levels of various free protein amino acids in the seeds. GABA-enriched rice grains were milled to a fine powder for oral administration to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Six weeks of administration showed that transgenic rice brings about a 20 mmHg decrease in blood pressure in two different kinds of SHRs, while there was no significant hypotensive effect in WKYs. These results suggest an alternative way to control and/or cure hypertension in humans with GABA-enriched rice as part of a common daily diet.     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

The cell death factor,cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells

An elicitor derived from the cell wall of rice blast fungus (Magnaporthe grisea) causes cell death in suspension cultured cells of rice (Oryza sativa L.). To elucidate the role of M. grisea elicitor on metabolic pathway of rice cells, we performed metabolite profiling using capillary electrophoresis-mass spectrometry (CE/MS). Treatment with M. grisea elicitor increased the amounts of antioxidants and free amino acids and decreased the amount of metabolites in the tricarboxylic acid (TCA) cycle. Lower ATP concentration caused aberrant energy charge, concurrently with reduced amount of NAD(P)H in elicitor treated cells. Among free amino acids detected in this study, the level of gamma-aminobutyric acid (GABA) increased. GABA is metabolized through a bypass pathway of the TCA cycle called GABA shunt, which is composed of glutamate decarboxylase (GAD), GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). While M. grisea elicitor negligibly affected GAD and SSADH, GABA-T activity significantly decreased. The decrease in GABA-T activity was recovered by NADPH oxidase inhibitor, which prevents cell death induced by M. grisea elicitor. Thus, GABA accumulation observed in rice cells under elicitor stress is partly associated with GABA-T activity.Key words: metabolome, Magnaporthe grisea, capillary electrophoresis, mass spectrometry, gamma-aminobutyric acid, GABA transaminase, Oryza sativa     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD⁺/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.     

引进巨胚稻与普通稻的米质和营养成分分析比较

对引进的巨胚稻进行引种栽培并对其米质和营养成分进行全面分析.结果表明,巨胚稻的胚重是普通水稻胚重的3～5倍,多项营养成分如蛋白质、钙、磷、钾、钠、锰、VE、VB6和膳食纤维等的含量比普通稻米要高得多,这个结果表明巨胚稻是加工婴幼儿食品非常好的原料.     

A key ABA hydrolase gene,OsABA8ox3 is involved in rice resistance to Nilaparvata lugens by affecting callose deposition

Abscisic acid (ABA) is an important plant hormone in regulating abiotic and biotic stresses. OsABA8ox3 is the key gene in ABA hydrolase genes, and plays an important role in controlling ABA level, but little is known in rice resistance to insects. We used rice osaba8ox3 T-DNA insertion mutant (knocking down the OsABA8ox3 gene) to elucidate rice resistance to the insect. There were obvious phenotype differences between the osaba8ox3 T-DNA insertion mutant and wild-type (WT), and the relative expression of synthetase genes in the osaba8ox3 mutant was higher, while the relative expression of hydrolase genes was lower than that of WT, respectively. The electrical penetration graph (EPG) recording indicated that the osaba8ox3 mutant had the less sucking phloem sap duration compared with WT, which indicated a significant increase in rice resistance to brown planthopper (Nilaparvata lugens; BPH). The callose deposition in the osaba8ox3 mutant increased by 60.39%, 52.2%, 26.6% and 31.7% than that of WT after BPH feeding for 0, 24, 48, and 72 h, respectively. These results showed OsABA8ox3 gene played an important role in rice resistance to BPH, and also provided new insights into the mechanism of callose deposition regulation in response to the piercing-sucking pest.