Studies on γ Globulin of Rice Embryo

All globulin components hitherto found in many species of seeds, α, β, γ and δ globulins, were identified in rice grain by ultracentrifugal experiments and gel-filtration chromatography. Among them, γ globulin was found to occur in high concentration in embryo and bran which were the most active parts in biological functions of rice grain. Then γ globulin was isolated from embryo by gel-filtration chromatography on a Sephadex G-200 column. Purified γ globulin was homogeneous in ultracentrifugal analysis and it was found to be insoluble in cold saline solution. On the other hand, α and β globulins were found to be more concentrated in endosperm with considerable heterogeneity.     

Studies on γ Globulin of Rice Embryo

An improved method has been described for the isolation and purification of γ globulin from rice embryo. The method involves the extraction with phosphate buffer, pH 7.0 and ionic strength 0.1, the fractionation in saline solution of ionic strength 0.31, the removal of nucleic acids by precipitation with ammonium sulfate and the gel filtration chromatography on a Sephadex G-200 column. Although the preparations exhibited homogeneous patterns in sedimentation analysis, the electrophoretic patterns on polyacrylamide gels at pH 8.35 and ionic strength 0.11 exhibited at least two components. Three major components, γ₁, γ₂ and γ₃ globulins, were isolated by ion exchange chromatography on a DEAE Sephadex A-50 column. These components were revealed to be homogeneous in electrophoresis as well as sedimentation. N-Terminal amino acid compositions have also been described.

The molecular weight of γ₁ globulin was determined as 2.0 × 10⁵ by the Archibald method, and the intrinsic viscosity, [η], and the sedimentation coefficient, s_{20, w}^°, were found to be 0.0424 dl/g and 7.26S respectively. These values indicated the large asymmetry of the protein. The protein was composed of 18 residues of hexose, 3 residues of pentose, 6 residues of hexosamine and 1751 residues of amino acids: Lys₅₈, His₄₇, Arg₁₄₈, Asp₁₂₆, Glu₂₇₃, Gly₁₆₁, Ala₁₄₄, Val₁₂₁, Leu₁₀₆, Ile₇₂, Pro₈₃, Ser₁₃₆, Thr₄₈, Hyp₆₈, Cys₁₇, Met₁₆, Tyr₄₄, Trp₈, Phe₇₅ and amide ammonia₁₆₃. The N-terminal amino acid analysis suggested that the protein was composed of ten subunits. The properties and the composition were discussed in comparison with those of the 7S globulin of soybean cotyledon.     

Studies on γ Globulin of Rice Embryo

The secondary structure of γ₁ globulin from rice embryo was investigated by means of optical rotatory dispersion, circular dichroism and infrared spectroscopy. The optical rotatory dispersion curve of the native γ₁ globulin gave a trough at 233mμ with a [m′]₂₃₃ value of ?2,100°, and the Moffitt-Yang plot gave the parameters of a₀= ?237 and b₀= ?20. These data suggest the presence of 3% helix and 38%β structure in the molecule. Circular dichroism exhibits a negative extremum at 218 mμ, giving a [θ]_R value of ?3,730, which suggests the presence of 16°β structure. Infrared spectrum of a thin film of γ₁ globulin showed absorption bands at 695 and 660 cm^?1 with a small hump at 615 cm^?1 characteristic of the β structure, random coil and α helix, respectively. The protein in heavy water exhibits the absorption maximum at 1,630cm^?1 which is also characteristic of the β structure.     

Studies on γ Globulin of Rice Embryo

The molecular weight of γ₃ globulin was determined to be 120,000 daltons by means of both sedimentation equilibrium and gel filtration methods. The protein was composed of 3 identical major and 1 minor subunits, and the molecular weights of them were found to be 35,000 and 13,000 daltons, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major subunit has an arginyl residue as the amino terminal amino acid. The amino acid and carbohydrate composition of γ₃ globulin was determined as follows: Lys₃₇His₃₇Arg₉₂Asp₆₀Glu₁₃₉Gly₁₂₀Ala₈₃Val₇₆Leu₇₀Ile₃₁Pro₅₄Ser₇₇Thr₃₃Cys₁₁Met₉Phe₅₁Tyr₂₆Trp₈ (Amide NH₃)₆₉Hexose₁₅Pentose₄Hexosamine₄. The structure of γ₃ globulin was discussed with comparing that of γ₁ globulin.     

Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Multiple Forms of Rice Seeds β-Amylases on Zymogram

Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.     

Morphological Observation on “Polyembryonic Seedling” of Rice

The formation of the so-called polyembryonic seedling and its morphology were observed. The main conclusions are as follows: 1. All caryopses contain only one embryo; 2, “The polyembryonic seedling” reported is actually single seedling with 1 or 2 lateral shoots sprouted from the main shoot. The first lateral shoot arises from the axil of coleoptile and the second one from the axil of lower leaf in the first lateral shoot. These lateral shoots are not independent seedlings. The formation of lateral shoot in wheat is the same as that of rice as mentioned above. The authors had dissected 1500 rice caryopses, 1600 young seedlings from the field and 1102 seedlings germinated under artificial conditions. Thus, polyembryonic seedlings as preriously reported is not present, at least, in rice C1001B line.     

Three Forms of α-Glucosidase from Suspension-cultured Rice Cells

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the three enzymes were inhibited by 11.25 ng of castanospermine. The inhibition was competitive.     

The Application of α-Picolinic Acid on the Screening of Rice Blast-Resistant Variants

The screening of rice (Oryza sativa L.) variants resistant to blast was studied by using α-picolinic acid treatment. In order to establish the selection conditions, anthers and anther-derived calla of rice strain C2, which is susceptible to blast, were cultured ma media supplemented with different concentrations of α-picolinic acid to examine the response to α-picolinic acid. it was shown that callus induction frequency of anther and callus growth were obviously declined in relevance to the increasing the concentrations of α-picolinic acid. 40 mg/L of α-picolinic acid was the crucial concentration for anther-derived callus induction and growth of the rice strain C2. The callus lines tolerated to 40 mg/L of α-picolinic acid were selected from rice strain C2 and then regenerated into green plants. By identification of blastresistance in the seedling and boot stage of R1 and R2 generations, two variants No. 4-091 and No. 2-07 resistant to Pyricularia oryzae strain ZA1 and ZB11 were obtained, however, the grain yield per plant of these variants were not so high as C2.     

Development of the “Third-Generation” Hybrid Rice in China

Rice is a major cereal crop for China. The development of the ‘‘three-line" hybrid rice system based on cytoplasmic male sterility in the 1970 s(first-generation) and the ‘‘two-line" hybrid rice system based on photoperiod-and thermo-sensitive genic male-sterile lines(second-generation)in the 1980 s has contributed significantly to rice yield increase and food security in China. Here we describe the development and implementation of the ‘‘third-generation" hybrid rice breeding system that is based on a transgenic approach to propagate and utilize stable recessive nuclear male sterile lines, and as such, the male sterile line and hybrid rice produced using such a system is nontransgenic. Such a system should overcome the intrinsic problems of the ‘‘first-generation" and‘‘second-generation" hybrid rice systems and hold great promise to further boost production of hybrid rice and other crops.     

Characterization and Use of Male Sterility in Hybrid Rice Breeding

The hybrid rice （Oryza sativa L.） breeding that was Initiated In China in the 1970s led to a great improvement in rice productivity. In general, It increases the grain yield by over 20% to the inbred rice varieties, and now hybrid rice has been widely introduced into Africa, Southern Asia and America. These hybrid varieties are generated through either three-line hybrid and two-line hybrid systems; the former is derived from cytoplasmic male sterility （CMS） and the latter derived from genlc male sterility （GMS）. There are three major types of CMS （HL, BT and WA） and two types of GMS （photoperlod-sensitlve （PGMS） and temperature-sensitive （TGMS））. The BT- and HL-type CMS genes are characterized as orf79 and orfH79, which are chimeric toxic genes derived from mltochondrial rearrangement. Rf3 for CMS-WA Is located on chromosome 1, while Rf1, Rf4, Rf5 and Rf6 correspond to CMS-BT, CMSoWA and CMS- HL, located on chromosome 10. The Rfl gene for BT-CMS has been cloned recently, and encodes a mltochondriatargeted PPR protein. PGMS Is thought to be controlled by two recessive loci on chromosomes 7 and 12, whereas nine recessive alleles have been identified for TGMS and mapped on different chromosomes. Attention Is still urgently needed to resolve the molecular complexity of male sterility to assist rice breeding.     

Cloning,Expression, and Characterization of Rice α-Galactosidase

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.     

Purification and Some Properties of Active β-Amylase in Rice

An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.     

Performance of Hybrids between Weedy Rice and Insect-resistant Transgenic Rice under Field Experiments： Implication for Environmental Biosafety Assessment

Transgene escape from genetically modified (GM) rice Into weedy rice via gene flow may cause undesired environmental consequences. Estimating the field performance of crop-weed hybrids will facilitate our understanding of potential introgression of crop genes (including transgenes) into weedy rice populations, allowing for effective biosafety assessment. Comparative studies of three weedy rice strains and their hybrids with two GM rice lines containing different insect-resistance transgenes (CpTl or BtlCpTI) indicated an enhanced relative performance of the crop-weed hybrids, with taller plants, more tillers, panicles, and spikelets per plant, as well as higher 1000-seed weight, compared with the weedy rice parents, although the hybrids produced less filled seeds per plant than their weedy parents. Seeds from the F1 hybrids had higher germination rates and produced more seedlings than the weedy parents, which correlated positively with 1000-seed weight. The crop-weed hybrids demonstrated a generally enhanced relative performance than their weedy rice parents in our field experiments. These findings indicate that transgenes from GM rice can persist to and introgress into weedy rice populations through recurrent crop-to-weed gene flow with the aid of slightly increased relative fitness in F1 hybrids.     

Variability in Aggressiveness of Rice Blast (Magnaporthe oryzae) Isolates Originating from Rice Leaves and Necks: A Case of Pathogen Specialization?

Rice blast, caused by Magnaporthe oryzae, causes yield losses associated with injuries on leaves and necks, the latter being in general far more important than the former. Many questions remain on the relationships between leaf and neck blast, including questions related to the population biology of the pathogen. Our objective was to test the hypothesis of adaptation of M. oryzae isolates to the type of organ they infect. To that aim, the components of aggressiveness of isolates originating from leaves and necks were measured. Infection efficiency, latent period, sporulation intensity, and lesion size were measured on both leaves and necks. Univariate and multivariate analyses indicated that isolates originating from leaves were less aggressive than isolates originating from necks, when aggressiveness components were measured on leaves as well as on necks, indicating that there is no specialization within the pathogen population with respect to the type of organ infected. This result suggests that the more aggressive isolates involved in epidemics on leaves during the vegetative stage of the crop cycle have a higher probability to infect necks, and that a population shift may occur during disease transmission from leaves to necks. Implications for disease management are discussed.     

Cloning and Characterization of AKR4C14, a Rice Aldo–Keto Reductase, from Thai Jasmine Rice

Aldo–keto reductase (AKR) is an enzyme superfamily whose members are involved in the metabolism of aldehydes/ketones. The AKR4 subfamily C (AKR4C) is a group of aldo–keto reductases that are found in plants. Some AKR4C(s) in dicot plants are capable of metabolizing reactive aldehydes whereas, such activities have not been reported for AKR4C(s) from monocot species. In this study, we have screened Indica rice genome for genes with significant homology to dicot AKR4C(s) and identified a cluster of putative AKR4C(s) located on the Indica rice chromosome I. The genes including OsI_04426, OsI_04428 and OsI_04429 were successfully cloned and sequenced by qRT-PCR from leaves of Thai Jasmine rice (KDML105). OsI_04428, later named AKR4C14, was chosen for further studies because it shares highest homology to the dicot AKR4C(s). The bacterially expressed recombinant protein of AKR4C14 was successfully produced as a MBP fusion protein and his-tagged protein. The recombinant AKR4C14 were capable of metabolizing sugars and reactive aldehydes i.e. methylglyoxal, a toxic by-product of the glycolysis pathway, glutaraldehyde, and trans-2-hexenal, a natural reactive 2-alkenal. AKR4C14 was highly expressed in green tissues, i.e. leaf sheets and stems, whereas flowers and roots had a significantly lower level of expression. These findings indicated that monocot AKR4C(s) can metabolize reactive aldehydes like the dicot AKR4C(s) and possibly play a role in detoxification mechanism of reactive aldehydes.