Giant Embryo Mutant Research Articles

A rice mutant with a giant embryo has increased levels of lipophilic antioxidants, E vitamers, and γ‐oryzanol fraction

AbstractBackground and objectiveLipophilic compounds in rice bran have health beneficial and functional properties. These lipophilic antioxidants in a giant embryo mutant (GEDrew) derived from a tropical japonica variety Drew were determined to evaluate its potential as a specialty variety.FindingsGEDrew had double the size of embryo and lower kernel weight than Drew. The majority of the tocols in the embryo were in the form of tocopherols with α‐tocopherol accounting for approximately 70%, while the tocopherols in bran ranged from 22% to 43% of total tocols. On a per embryo basis, GEDrew had a higher quantity of tocols compared with Drew; however, on a weight basis, the concentration of tocols in GEDrew was less than Drew. The γ‐oryzanol concentration in embryos was the same for both the mutant and wild type. The smaller brown rice kernel size and bigger embryo size of GEDrew resulted in higher proportion of bran + embryo (w/w), which led to higher total tocopherols, tocotrienols, and γ‐oryzanol in the whole grain of GEDrew relative to Drew.ConclusionA rice mutant with a large embryo had an elevated level of γ‐oryzanol and tocols, both as a group and as individual homologs, in whole grains as compared to the wild type.Significance and noveltyGEDrew has the potential to be grown as a specialty rice variety for the purposes of isolating its bran, oil, and lipophilic antioxidants for use by the food and cosmetic industries.

Antihypertensive effect of giant embryo brown rice and pre-germinated giant embryo brown rice on spontaneously hypertensive rats.

“Shangshida NO.5” is a giant embryo mutant resulting from giant embryo gene (GE) dysfunction in “Chao2‐10” rice. Here, we compared the antihypertensive effects of “Chao2‐10” brown rice (C2‐10), “Shangshida NO.5” brown rice (GER), and pre‐germinated “Shangshida NO.5” brown rice (PGER) in spontaneously hypertensive rats (SHR). Male SHR at 6 weeks of age were divided into four groups and were fed with (a) a control diet (control), (b) a 40% C2‐10‐supplemented diet (C2‐10), (c) a 40% GER‐supplemented diet (GER), or (d) a 40% PGER‐supplemented diet (PGER) for 8 weeks, and their physiological and biochemical parameters were measured. The results showed that the C2‐10‐, GER‐, and PGER‐supplemented diets significantly decreased systolic blood pressure (SBP) and diastolic blood pressure (DBP) during the experiment. At the end of the experimental period, the SBP and DBP of the C2‐10, GER, and PGER groups were 7.6, 23.3, and 31.1 mmHg and 9.8, 21.1, and 29 mmHg lower than those in the control group, respectively, suggesting the GER and PGER diets were better able to inhibit blood pressure elevation than the C2‐10 diet. The serum creatinine levels in the C2‐10, GER, and PGER groups and the blood urea nitrogen content in the PGER group were significantly lower than those of the control group, indicating that C2‐10‐, GER‐, and especially PGER‐supplemented diets improved renal function. In addition, the antioxidant activity of the C2‐10 group and especially of the GER and PGER groups also improved. The above results suggest that “Shangshida NO.5” rice, particularly pre‐germinated rice, is a good dietary supplement for preventing the development of hypertension.

Identification and Characterization of LARGE EMBRYO, a New Gene Controlling Embryo Size in Rice (Oryza sativa L.)

BackgroundAlthough embryo accounts for only 2–3% of the total weight of a rice grain, it is a good source of various nutrients for human health. Because enlarged embryo size causes increase of the amount of nutrients and bioactive compounds stored within rice grain, giant embryo mutants of rice (Oryza sativa L.) are excellent genetic resources for improving the nutritional value of rice grains.ResultsThree giant embryo mutants, including large embryo (le), giant embryo (ge) and super-giant embryo (ges), with variable embryo size were used in this study. We investigated whether genes controlling embryo size in these mutants (le, ge and ges) were allelic to each other. Although ge and ges was allelic to GIANT EMBRY (GE), le was not allelic to ge and ges in allelism test. The GE gene carried a unique nucleotide substitution in each of the two mutants (ge and ges), resulting in non-synonymous mutations in exon 2 of GE in both mutants. However, the GE gene of the le mutant did not carry any mutation, suggesting that the enlarged embryo phenotype of le was governed by another gene. Using map-based cloning, we mapped the LE gene to the short arm of chromosome 3. The le mutant showed mild enlargement in embryo size, which resulted from an increase in the size of scutellar parenchyma cells. The LE encodes a C3HC4-type RING finger protein and was expressed to relatively high levels in seeds at a late developmental stage. Knockdown of LE expression using RNA interference increased the embryo size of rice grains, confirming the role of LE in determining the embryo size.ConclusionOverall, we identified a new gene controlling embryo size in rice. Phenotypic and molecular characterization results suggest that the le mutant will serve as a valuable resource for developing new rice cultivars with large embryos and nutrient-dense grains.

Barley lys3 mutants are unique amongst shrunken-endosperm mutants in having abnormally large embryos

Many shrunken endosperm mutants of barley (Hordeum vulgare L.) have been described and several of these are known to have lesions in starch biosynthesis genes. Here we confirm that one type of shrunken endosperm mutant, lys3 (so called because it was first identified as a high-lysine mutant) has an additional phenotype: as well as shrunken endosperm it also has enlarged embryos. The lys3 embryos have a dry weight that is 50–150% larger than normal. Observations of developing lys3 embryos suggest that they undergo a form of premature germination and the mature lys3 grains show reduced dormancy. In many respects, the phenotype of barley lys3 is similar to that of rice GIANT EMBRYO mutants (affected in the OsGE gene). However, the barley orthologue of OsGE is located on a different chromosome from Lys3. Together these results suggest that the gene underlying Lys3 is unlikely to encode a starch biosynthesis protein but rather a protein influencing grain development.

Development and evaluation of rice giant embryo mutants for high oil content originated from a high-yielding cultivar 'Mizuhochikara'.

Rice bran oil is a byproduct of the milling of rice (Oryza sativa L.). It offers various health benefits and has a beneficial fatty acid composition. To increase the amount of rice bran as a sink for triacylglycerol (TAG), we developed and characterized new breeding materials with giant embryos. To induce mutants, we treated fertilized egg cells of the high-yielding cultivar ‘Mizuhochikara’ with N-methyl-N-nitrosourea (MNU). By screening M2 seeds, we isolated four giant embryo mutant lines. Genetic analysis revealed that the causative loci in lines MGE12 and MGE13 were allelic to giant embryo (ge) on chromosome 7, and had base changes in the causal gene Os07g0603700. On the other hand, the causative loci in lines MGE8 and MGE14 were not allelic to ge, and both were newly mapped on chromosome 3. The TAG contents of all four mutant lines increased relative to their wild type, ‘Mizuhochikara’. MGE13 was agronomically similar to ‘Mizuhochikara’ and would be useful for breeding for improved oil content.

Effect of instant cooked giant embryonic rice on body fat weight and plasma lipid profile in high fat-fed mice.

The comparative effects of instant cooked rice made from giant embryo mutant or ordinary normal rice on body weight and lipid profile in high fat-fed mice were investigated. The animals were given experimental diets for seven weeks: normal control (NC), high fat (HF), and HF supplemented with instant normal white (HF-NW), normal brown (HF-NB), giant embryonic white (HF-GW), or giant embryonic brown (HF-GB) rice. The HF group showed markedly higher body weight, body fat, plasma and hepatic triglyceride and cholesterol concentrations, and atherogenic index relative to NC group. However, instant rice supplementation counteracted this high fat-induced hyperlipidemia through regulation of lipogenesis and adipokine production. The GB rice exhibited greater hypolipidemic and body fat-lowering effects than the GW or NB rice. These findings illustrate that the giant embryo mutant may be useful as functional biomaterial for the development of instant rice with strong preventive action against high fat diet-induced hyperlipidemia and obesity.

Loss of Function of the Cytochrome P450 Gene CYP78B5 Causes Giant Embryos in Rice

Rice (Oryza sativa L.) is one of the most important food crops in the world. Most kernel oil and micro-nutrients are located in the embryo whereas starch is mainly in the endosperm. Although embryo size is important for rice grain yield and food processing, little is known about the genetic mechanisms controlling embryo size. We characterized a rice giant embryo mutant, giant embryo (ge), which was derived from wild-type Kinmaze by chemical mutagenesis. The large embryo size resulted from larger cell size. The gene controlling the giant embryo trait was cloned by a map-based approach, and shown to encode cytochrome P450 protein CYP78B5, which was previously designated as CYP78A13. Gain-of-function transgenic experiments on OsGE/CYP78B5 in the ge mutant restored the normal seed phenotype. Expression analysis showed that the OsGE/CYP78B5 gene product accumulated mainly in young developing seeds. Subcellular localization indicated that OsGE/CYP78B5 resides in the peroxisomes, and the N-terminal 51 amino acid residues function as a targeting signal. The IAA levels were one-third those of wild type in GE mutants at 6 days after fertilization, yet the embryonic cells were much larger. OsGE/CYP78B5 also regulates expression levels of auxin-responsive and cyclin-related genes. Isolation of GE provides an opportunity to manipulate the nutritive composition and distribution of the grain by marker-assisted selection and transgenic approaches.

Hypolipidemic and Body Fat–Lowering Effects of Giant Embryo Brown Rice (Seonong 17 and Keunnunjami) in High‐Fat‐Fed Mice

ABSTRACTThe effects of giant embryo brown rice Seonong 17 and Keunnunjami, in comparison with that of ordinary normal brown rice, on the body weight and lipid profile in mice under a high‐fat diet condition were investigated. The animals were fed with experimental diets for eight weeks: normal control, high fat (HF), and high fat supplemented with normal brown rice, Seonong 17 rice, or Keunnunjami rice powder. Their body and fat weights, lipid profiles, lipogenic enzyme activities, and adipokine concentrations were determined. Significant differences between the animal groups were analyzed with Tukey's test (P < 0.05). A marked increase in the body weight and fat, plasma triglyceride and total cholesterol concentrations, and free fatty acid level were found in HF mice relative to the control group. On the other hand, diet supplementation with rice, particularly the giant embryo rice samples, significantly suppressed this high‐fat‐induced body weight gain and hyperlipidemia via regulation of lipogenesis and adipokine production. Both the Seonong 17 and Keunnunjami exhibited greater hypolipidemic and body fat–lowering effects than the normal brown rice. The results demonstrate that these giant embryo mutants may be beneficial as biomaterials for functional foods with antiobesity and hypolipidemic effects.

Hypoglycemic and Antioxidative Effects of Instant Cooked Giant Embryonic Rice in High‐Fat‐Fed Mice

ABSTRACTThe effect of instant cooked rice made from giant embryo mutant or ordinary normal rice on the glucose metabolism and antioxidative defense system in mice under a high‐fat (HF) diet condition was investigated. The animals were randomly divided and given experimental diets for seven weeks: normal control diet, HF diet, and HF diet supplemented with instant normal white, normal brown, giant embryonic white, or giant embryonic brown rice. At the end of the experimental period, the HF mice showed a marked increase in the blood glucose level, insulin concentration, and plasma and erythrocyte lipid peroxidation and a significant decrease in the glycogen level relative to the control group. However, diet supplementation with instant cooked giant embryonic and normal brown rice counteracted this high‐fat‐induced hyperglycemia and oxidative stress through regulation of the glucose‐regulating and antioxidant enzyme activities. The giant embryonic brown rice was the most effective in improving the glucose metabolism and antioxidant defense status in mice under the HF diet condition. The results demonstrate that the giant embryo rice mutant may be useful in the development of instant cooked rice with strong hypoglycemic and antioxidative properties.

Physicochemical Properties of a Giant Embryo Mutant Induced by T-DNA Insertion in Rice

This study was conducted to determine the physicochemical properties of a giant embryo rice ‘47JB-4-B-5-B’derived from the cross between ‘47’ a mutant of ‘wayoung’induced by T-DNA insertion, and ‘unam’ The grain appearance and chemical components of the embryo were analyzed and compared with a donor cultivar, ‘wayoung’ The proportion of embryo weight to grain weight of ‘47 JB-4-B-5-B’was 2.2 times heavier (6.7%) than that (3.1%) of ‘wayoung’ Total free amino acid content (75.81 mg/100 g) of ‘47JB-4-B-5-B’was 2.1 times higher than that of ‘wayoung’ The GABA content in brown rice was 14.06 mg/100 g in ‘47JB-4-B-5-B’and 6.8 mg/100 g in ‘wayoung’ Especially, the GABA content in brown rice of ‘47JB-4-B-5-B’remarkably increased (about 33 times from 1.48 mg to 44.81 mg/100 g) 2 days after germination. Continuous frequency distributions and transgressive segregation in embryo length and width were observed in the F 2 population of the cross between ‘47’and ‘heongcheong’ indicating that the giant embryo was controlled by quantitative trait loci. However, embryo length and width demonstrated high broad sense heritability, implying that giant embryonic traits could be selected in earlier generations in comparison with other quantitative traits.

γ-Oryzanol, tocol and mineral compositions in different grain fractions of giant embryo rice mutants

Rice embryo is concentrated with lipid, protein and some bioactive chemicals. Two rice mutants IR64-GE and TNG71-GE (M7 generation) were characterised by an enlarged embryo compared with their wild types. In the present study, distributions of protein, lipid, total phenolics, γ-oryzanol, tocols and some essential minerals in these two giant embryo mutants and their respective normal embryo wild types IR64 and TNG71 were compared. The embryo dry weights of giant embryo mutants IR64-GE and TNG71-GE were 0.92 and 1.32 mg per seed respectively. These values were higher than those of their respective normal embryo genotypes (0.50 and 0.62 mg per seed). Large variations in protein, lipid, phenolic, γ-oryzanol, tocol and minerals levels were found between mutant and wild-type pairs. The brown rice of TNG71-GE had higher total γ-oryzanol (average of 24% increase) and total tocol (average of 75% increase) levels than TNG71, IR64 and IR64-GE. The embryo and bran parts of giant embryo mutant TNG71-GE were found to be good sources of vitamin E and γ-oryzanol. Therefore it could be used to produce high-value by-products from milled embryo and bran parts and as a genetic resource for rice improvement programmes. TNG71-GE can also be used as a nutrient-fortified rice cultivar.

Comparative analysis of physicochemicals and antioxidative properties of new giant embryo mutant, YR23517Acp79, in rice (Oryza sativa L.)

Nutritional and physicochemical properties of new giant embryo mutant rice (YR23517Acp79, YR) were analyzed. YR exhibited increased total protein (9.3±0.3%), lipid (3.7±1.1%), amino acid (663.28±1.9 mg/g), and mineral contents (Ca=284.0±6.2, Mg=1417.5±13.6 mg/kg). In YR brown rice four major physicochemicals, including gamma-aminobutyric acid (brown rice= 0.46±0.014 mg/g), γ-oryzanol (0.43±0.021 mg/g), vitamin B1 (6.42±0.3 mg/kg), and tocopherols (alpha= 2.68±0.1, beta=0.11±0.01, gamma=0.05±0.001 mg/100 g) increased in comparison to reported giant embryo (Keunnunbyeo, KB) and normal embryo rice (Ilmibyeo, IB). YR showed higher scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (0.2 g/mL=57.1±2.25) and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (0.2 g/mL=50.2±1.45) radicals but also inhibited lipopolysaccharide-induced nitric oxide production without cytotoxicity. These results indicate YR is a high quality functional rice due to its high nutrition content and antioxidant effects of physicochemicals.

Molecular characterization and physico-chemical analysis of a new giant embryo mutant allele (ge t) in rice (Oryza sativa L.)

The rice embryo is rich in lipid and protein bodies, bioactive chemicals such as dietary fiber, phytic acids, vitamin B and E, and gamma aminobutyric acid (GABA) than the endosperm. In this paper, we report a new giant embryo mutant,get, induced from somaclonal variation derived by anther culture in rice. Sequence analysis of Cytochrome P450 of the get mutant revealed thatget is a new allele of theGE gene with a single point mutation with substitution of amino acid, W395 to L395. The weight of theget mutant embryo was 3.7 times higher than normal embryo. Tocopherol and mineral content were also higher than the previously reported giant embryo rice variety, Keunnun. These results indicated that this new giant embryo rice (get) offers a promising source of genetic material in improving nutritional quality of rice especially tocopherol, essential minerals, and GABA.

Characterization of indica-type giant embryo mutant rice enriched with nutritional components

Five giant embryo mutants, described as MH-gel, MH-ge2, MH-ge3, MH-ge4 and MH-ge5, which were derived from the same indica rice cv. ‘Minghui 86’ and characterized by 2.0, 1.88, 2.08, 1.93 and 1.88 times enlarged embryo than that of wild type, were selected for the current study. The mutated giant embryos were controlled by a single recessive gene, and except mutated locus with MH-ge1 other four loci were allelic to each other and the previous reported locus ge in japonica rice cv. ‘Kinmaze’. No obvious differences in physicochemical properties such as apparent amylose content (AAC), alkali spreading value (ASV), gel consistency (GC), and starch paste viscosity were observed between giant embryo mutants and wild type. Significant increases in the contents of crude lipid (LC), crude protein (PC), Vitamin B1 (VB1), Vitamin B2 (VB2), Vitamin E (VE), essential amino acids such as Arginine (Arg), Aspartic acid (Asp), Glutamic acid (Glu), Lysine (Lys), Methionine (Met), and mineral elements such as calcium (Ca), iron (Fe), potassium (K), phosphorus (P) and zinc (Zn) were detected in brown rice (BR) of giant embryo mutants. The amounts of gamma aminobutyric acid (GABA), an inhibitory neurotransmitter, were similar in the BR of giant embryo mutants and wild type, and more GABA content was observed in germinated brown rice (GBR) than BR. Significant enrichments were detected in the GBR of giant embryo mutants, basically corresponding to the enlarged embryo.

How is embryo size genetically regulated in rice?

It is unclear how embryo size is genetically regulated in plants. Since cereals have a large persisting endosperm, it is expected that embryo size is affected by endosperm development. Nine single recessive mutations, four reduced embryo mutations representing three loci, REDUCED EMBRYO1, REDUCED EMBRYO2 and REDUCED EMBRYO3, four giant embryo mutations derived from a single locus GIANT EMBRYO, and one endospermless mutation endospermless1-2 were analyzed. Every reduced embryo mutation caused reduction of all the embryonic organs including apical meristems and the enlargement of the endosperm. The giant embryo mutants have a reduced endosperm and an enlarged scutellum. However, shoot and radicle sizes were not affected. All the reduced embryo and giant embryo mutations did not largely affect postembryonic development. Accordingly, the expression of genes analyzed are seed-specific. In reduced embryo and giant embryo mutations, abnormalities were detected in both embryo and endosperm as early as 2 days after pollination. endospermless1-1 resulted in an early loss of endosperm, yielding a giant embryo, suggesting that embryo growth was physically limited by the endosperm. A double mutant between giant embryo-2 and club-shaped embryo1-1, which has a normal endosperm and a minute undifferentiated embryo, resulted in a club-shaped embryo1-1 embryo and a reduced endosperm of giant embryo-2, indicating that GIANT EMBRYO regulates the endosperm development. Double mutants between giant embryo-2 and three reduced embryo mutants exhibited the reduced embryo phenotype in both embryo and endosperm, suggesting that reduced embryo mutations cause the enlarged endosperm. Further, a double mutant of reduced embryo3 and endospermless1-1 showed the enlarged embryo in endospermless seed. This confirms that reduced embryo3 does not regulate embryo size but enlarges endosperm size. Together with the results of the other double mutant analysis, REDUCED EMBRYO1, REDUCED EMBRYO2, REDUCED EMBRYO3 and GIANT EMBRYO are concluded to regulate endosperm development.

Phenotypic diversity of 188 rice embryo mutants

AbstractWe have identified 188 embryo mutants of rice and characterized them into six groups based on their phenotypes: (1) embryoless in mature seed, (2) deletion of embryonic organ(s), (3) abnormal position of embryonic organs, (4) abnormal embryo size, (5) defect in organ morphology, and (6) variable abnormal phenotypes in spite of single mutations. Three types of organless mutants are obtained: small globular embryo, club‐shaped embryo, and large embryo. Although 12 shootless mutants derived from at least three loci are identified, only three radicleless mutants are recovered, which produce normal adventitious roots after germination. In reduced embryo mutants, every embryonic organ is reduced, in contrast to giant embryo mutants in which only scutellum is enlarged. Considerable number of mutants are categorized into (5) and (6) in the above. These diverse embryo mutants would serve as promising materials for genetic study of embryogenesis. © 1995 Wiley‐Liss, Inc.

Oil content and fatty acid composition of a giant embryo mutant in rice.

油飼料イネ育種のための基礎研究として巨大胚突然変異系統(EM-40)と正常型(全南風)において，胚，糊粉層および澱粉層の含油量と脂肪酸組成を比較検討した. 玄米千粒重について巨大胚突然変異系統は20.18gと正常型の22.28gより軽かった.しかし，胚は長さ，幅が大きく，胚重が2.6倍に達した.一方糊粉層はやや厚くなったにもかかわらず，糊粉層重はほぼ同じであった. 正常型では，含油量0，589のうち，胚と糊粉層いわゆる糠で74劣，巨大胚突然変異系統では含油量O.79gのうち糠が83%を占めていた.巨大胚突然変異系統の玄米の含油量が，正常型に比べ，1.36倍に増加したことは，巨大胚の胚中の含油量が正常型の胚の1.94倍，糊粉層で1.30倍に増加Lたことに起因していた.玄米における含曲率は，正常型が2.60%で，巨大胚では3，91%と高くなっていた.しかし，正常型の胚の含油率の29.09%に比較して巨大胚突然変異系統は21・09%に減少Lていた・これは胚の巨大化により，胚中の滴の充実が低下したためと考えられる.一方糊粉層の含油率は正常型の1.36倍に増加していた.澱粉層の含曲率は，正常型，巨大胚突然変異系統ともほとんど変らず1%以下であった. 正常型の脂肪酸組成は，胚，糊粉層および澱粉層ともほとんど変らなかったが，巨大胚突然変異系統では，胚の巨大化に伴ってステアリン酸がやや増加していた.