Antioxidant,anti-amylase and anti-glycation potential of brans of some Sri Lankan traditional and improved rice (Oryza sativa L.) varieties

Brans of 23 traditional and 12 improved (both red and white) rice varieties in Sri Lanka were screened for anti-amylase and anti-glycation activities in vitro. Varieties which showed the highest inhibitory activities at screening were further investigated for anti-glucosidase and glycation reversing as anti-diabetic properties. The same varieties were studied for selected antioxidant properties. Significantly high anti-amylase and anti-glycation activities were observed for bran extracts of red varieties compared to white varieties at screening. Traditional red rice varieties, Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti, exhibited significant and dose dependent anti-amylase, anti-glycation and glycation reversing activities. These varieties also showed marked antioxidant properties. It is concluded that brans of Sri Lankan traditional red rice varieties Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti may be potential food supplements for diabetes.     

Metabolite profiling of colored rice (Oryza sativa L.) grains

HPLC analysis of dehulled red, black and non-colored indica and japonica rice subspecies revealed significant differences in the contents of the anthocyanins cyanidin-3-glucoside and peonidin-3-glucoside. The rice materials were subjected to a comparative capillary gas chromatography-based metabolite profiling approach. The employed extraction and fractionation protocol enables the analysis of a broad spectrum of lipophilic and hydrophilic low molecular weight constituents from different chemical classes. The method covers not only primary metabolites (e.g. sugars, fatty acids) but also nutritionally relevant constituents (e.g. α-tocopherol, γ-aminobutyric acid). Statistical assessment of the data via principal component analysis and agglomerative hierarchical clustering revealed a distinct grouping of the different colored rice subspecies on the basis of their metabolite profiles. Compared to non-colored and red rice, black rice exhibited, in particular, higher levels of fatty acid methyl esters, free fatty acids, organic acids and amino acids.     

Effects of winter flooding on weedy rice (Oryza sativa L.)

Weedy rice (Oryza sativa L.), characterised by competitiveness, seed longevity, and dormancy is a troublesome weed to rice fields. Furthermore, its close botanical affinity to cultivated rice makes its control particularly difficult. However, winter flooding of rice fields can be an efficient technique to control weedy rice infestation by promoting weed seed decay, animal predation, or germination.     

Agronomic,molecular and antioxidative characterization of red- and purple-pericarp rice (Oryza sativa L.) mutants in Taiwan

Pigmentation of rice grain is controlled by Ra, Rc and Rd genes, and the expressions of these genes differ among red-, purple- and white-pericarp varieties. The present study examined the grain yield and the expression of Ra, Rc and Rd genes in non-waxy white-pericarp rice SA418 and waxy white-pericarp rice SA419 and their respective red-pericarp and purple-pericarp mutants with waxy or non-waxy endosperm. Significant variations in 100-grains weight and grain yield were observed among the tested mutants. The Ra was expressed in purple-pericarp mutants, while the Rc was expressed in red-pericarp mutants. The total phenolics, total flavonoids, total anthocyanins and total proanthocyanidins contents and antioxidant activities in the bran part also differed among the mutants. Non-waxy red-pericarp mutant M-69 had heavier 100-grains weight (2.86 g), contained more total phenolics (49.37 mg g⁻¹ bran dry weight) and produced higher grain yield (6.93 t ha⁻¹) than white-pericarp rices SA418 (2.43 g, 2.89 mg g⁻¹ bran dry weight and 2.80 t ha⁻¹, respectively) and SA419 (2.62 g, 2.20 mg g⁻¹ bran dry weight and 6.73 t ha⁻¹, respectively). Thus, the polished rice grain of M-69 can be used for staple food consumption, and its bran parts are useful for producing health-promoting by-product.     

Differential tolerance of weedy red rice (Oryza sativa L.) from Arkansas, USA to glyphosate

Red rice (Oryza sativa L.), a weedy relative of cultivated rice (O. sativa), is a troublesome weed in rice in the southern United States. It is also very problematic in other world regions where rice is grown. There are many types of red rice. Some have white pericarp. In this paper, the term ‘red rice’ will be used to refer to the weedy types that occur in this region of the southern USA. Because of the great diversity in morphology and phenology of red rice populations in the USA, we hypothesized that these have differential tolerance to glyphosate. Red rice is subjected to selection pressure by glyphosate, which is the principal herbicide used in glyphosate-resistant soybean planted after rice. Two hundred-fifteen red rice accessions were collected in Arkansas, USA between 2002 and 2003 and evaluated for glyphosate tolerance between 2003 and 2006. The commercial dose of glyphosate controlled red rice accessions 81-100% regardless of application timing, by 30 d after treatment. The number of survivors that produced seeds and the seeds produced per survivor differed between accessions. Application of the full dose at the tillering stage was more effective than at the 3- to 4-leaf stages. At half the field dose, 40 to 54% of survivors produced seed, with germination capacity of 30-100%. The lowest dose of glyphosate, 225 g a.e. ha⁻¹, resulted in the widest range of control ratings from 30 to 100%. The red rice accessions differentiated into six tolerance categories. A tolerance map was generated and will be used in educating growers about the proper use of glyphosate for red rice management.     

Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25 kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0–8.5 and at 45 °C with K_m and V_max values for 1-naphthyl acetate of 172 μM and 63.7 μmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.     

Diversity of weedy red rice (Oryza sativa L.) in Arkansas,U.S.A. in relation to weed management

Weedy rice, specifically red rice (Oryza sativa L.), is a major weed in rice which causes up to 80% yield loss and reduction of grain quality. Red rice accessions from Arkansas, U.S.A., were characterized to classify red rice accessions into certain phenotypic groups relevant to weedy rice management. The red rice accessions were 70% strawhull, 22% blackhull, 7% brownhull and <1% goldhull. Generally, blackhull red rice was the tallest (139 cm) and strawhull the shortest (133 cm) among all accessions. Blackhull red rice had more tillers (102/plant), smaller flag leaves (13 cm wide, 34 cm long), and flowered later (1225 heat units) than strawhull red rice which had 85 tillers/plant, 15 cm-wide and 34 cm-long flag leaves, and flowered after accumulating 1195 heat units. Morphological differences between accessions within each hull type were highly significant, showing great diversity within a hull color group as indicated by large ranges in traits. For example, blackhulls were 75–190 cm tall with 18–69 cm long flag leaves, 21–188 tillers and produced 40–949 g seed. Strawhulls were 46–189 cm tall with 18–66 cm flag leaf length, 16–172 tillers and produced 100–608 g seed. Some traits, such as seed production, differed widely between accessions within each hull color group such that the average seed production/accession for blackhull did not differ from that of strawhull weedy rice (196 vs. 192 g/plant). The onset of flowering among all accessions ranged from 56 to 126 d after planting. Red rice accessions formed six phenotypic clusters generally segregated by plant size or flowering time. Each morphotype would have different competitive abilities; thus, weedy rice management could be geared toward plant types. Highly competitive plant types would require intensive control measures to minimize yield losses and reduce the soil seed bank. Flowering dates impact stewardship strategies for herbicide-resistant or any genetically modified rice.     

Root growth dynamics and stomatal behaviour of rice (Oryza sativa L.) grown under aerobic and flooded conditions

Aerobic rice culture is a new technology designed to reduce water use, but the vulnerability of rice to aerobic condition has limited its development. The objective of this study was to characterize the root growth and stomatal behaviour of four rice cultivars grown in flooded and aerobic culture for 2 years. In aerobic culture, where the soil water potential at 20-cm depth averaged between −15 and −30 kPa, total root biomass was significantly lower than in flooded culture for the whole growth period, owing to a reduction in root biomass in the surface layer. Dry-matter partitioning to roots decreased, but the ratio of deep root biomass to total root biomass tended to be higher in aerobic culture than in flooded culture. The low root-to-shoot ratio and poor root growth in the surface layer in aerobic culture are attributable to the considerable reduction in adventitious root number. As a result, the varietal difference in total root biomass was due largely to individual root growth in aerobic culture. Stomatal closure was distinct at the vegetative stage in aerobic culture, even when the soil water potential was near field capacity, partly because of the poor rooting vigour. When the soil water potential at 20-cm depth was below −50 kPa, the stomatal behaviour reflected the root growth in the subsurface layer. These results suggest the role of vigorous root growth in soil water uptake and hence, in maintaining transpiration in aerobic rice culture.     

Identification of deletion mutants with improved performance under water-limited environments in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a semi-aquatic member of the grass family that is poorly adapted to dry environments and has greater sensitivity to water-deficits than other important cereals in this family. To increase productivity in aerobic or water-limited environments rice must overcome its adaptations to flooded environments. Deletion mutants offer an alternative genetic resource for improving drought tolerance. Almost 3500 IR64 deletion mutants were screened under vegetative and reproductive stage drought stress in the field and evaluated for leaf drying and/or grain yield. Seven novel conditional mutants of rice which showed gain of function through continued growth as drought stress developed compared to the wild type were identified. Mutant recovery rate was 0.1%. Further evaluation of putative drought mutants revealed that their average shoot biomass at maturity and grain yield per plant under stress exceeded those of the wild type by two-fold. Studies under controlled conditions confirmed mutants to have continued growth of both roots and shoots as drought developed compared to the wild type, and a tendency for greater water extraction. We propose that deletions in these mutants have affected a regulator of the highly conservative growth response common to irrigated lowland rice cultivars. Our results suggest that screening deletion mutants for performance under managed drought stress in the field could be a highly effective way to identify valuable genetic resources for improved drought response and aerobic adaptation in rice.     

Identification of novel glutelin subunits and a comparison of glutelin composition between japonica and indica rice (Oryza sativa L.)

Glutelin, a major protein in rice grains, is encoded by a multigene family. However, its protein composition is not well characterised. Here, we identified and characterised two novel glutelin subunits, GluBX and GluC. The individual glutelin subunits of japonica cv. Nipponbare and indica cv. 93-11 rice were analysed using 2-dimensional gel electrophoresis, LC–MS/MS, and Western blotting. Comparison of the glutelin profiles between three japonica and three indica cultivars indicated two distinct subunits (GluA-1 and GluA-3 isomers) and a distinction in the subunit composition (notably GluA-3 and Lys-rich GluB-1 components) of these two subspecies. Sequence alignment revealed different nutritional (Lys residues) and functional (Cys residues) characteristics between the type-A and type-B glutelin subfamilies. We also analysed amino acid and total protein contents of the grains in thirty-five cultivars, and we demonstrated that the Lys-rich glutelin composition of indica cultivars is superior to that of japonica cultivars. The Lys-rich and Cys-poor GluBX subunit is a native protein and is a high nutritional protein in grains. Our combined approaches for the identification of glutelin subunits have revealed the nutritional characteristics of individual subunits in rice, and this knowledge will provide new insights for improving grain quality during rice breeding.     

Nitrogen use efficiency in selected rice (Oryza sativa L.) genotypes under different water regimes and nitrogen levels

Water and nutrient availability are two major constraints in most rice-based rainfed shallow lowland systems of Asia. Both stresses interact and contribute to the low productivity and widespread poverty in this environment. The objective of this study was to improve the understanding of interaction between the two factors and to identify varietal characteristics beneficial for productivity in a water- and nutrient-limited rice environment. For this purpose, we screened 19 rice genotypes adapted to different rice environments under two water and two nutrient treatments during the wet season of 2004 and 2005 in southern Luzon, Philippines. Across all genotypes tested and in comparison with the irrigated control, rainfed conditions reduced grain yield of the treatment without N application by 69% in 2004 and by 59% in 2005. The mean nitrogen fertilizer response was highest in the dryer season of 2004 and the rainfed treatment, indicating that water stress had no effect on fertilizer response. Nitrogen application reduced the relative yield loss to 49% of the irrigated treatment in 2004 and to 52% of the irrigated treatment in 2005. Internal efficiency of N (IEN) and recovery efficiency of applied N (REN) were significantly different between genotypes, but were not affected by water availability (REN) or by water and nutrient availability (IEN). In contrast, grain yield and total N uptake were affected by cultivar, N and water availability. Therefore, germplasm for rainfed environments should be screened under conditions of limited and good nitrogen and water supplies. The four best cultivars, CT6510-24-1-2, IR55423-01, IR72, and IR57514-PMI5-B-1-2, performed well across all treatments and both years. Except for IR72, they were all characterized by medium height, medium duration, high early vigor, and a moderate level of drought tolerance. This combination of characteristics seems to enable the optimal use of limited water and nutrient resources occurring in many shallow rainfed lowlands. We also concluded that moderate drought stress does not necessarily affect the response to moderate N rates, provided that drought does not induce high spikelet sterility and that fertilizer N is properly managed.     

QTL mapping for crude protein and protein fraction contents in rice (Oryza sativa L.)

Improvement of rice storage proteins is important in rice breeding for high nutritional quality. Seventy-one recombinant inbred lines (RIL) derived from a cross between japonica variety Asominori and indica variety IR24 were used to study the inheritance of crude protein and protein fraction contents in rice. A total of 16 QTL were identified and mapped on eight chromosomes. Several QTL affecting contents of different protein fractions were mapped in the same chromosomal region. In particular, two QTL with a significant contribution were identified to simultaneously affect prolamin and glutelin contents. One QTL denoted as qCP-12 affecting crude protein content (CP) was located in the same region as QTL qGLT-12 affecting glutelin content, in agreement with the positive correlation between glutelin level and protein content. QTL with larger genetic effects were further confirmed using two sets of chromosome segment substitution lines (CSSL), where Asominori and IR24 were used as the recurrent parents. By QTL comparative analysis, two QTL for CP, three for globulin content and one for prolamin content were located in the vicinity of CP QTL previously identified in polished rice. Application of these results in rice breeding is discussed.     

Potential of Waxy gene microsatellite and single-nucleotide polymorphisms to develop japonica varieties with desired amylose levels in rice (Oryza sativa L.)

The Waxy (Wx) gene is responsible for the synthesis of amylose, a key determinant of the cooking and processing qualities of rice. Polymorphisms of CT-microsatellite and G–T single-nucleotide polymorphism (SNP) in the Wx gene and their relationship to amylose content (Ac) were explored using 178 non-waxy rice genotypes. Nine Wx microsatellite alleles, namely (CT)10 and 11, and (CT)14–20 were identified and 11 haplotypes were recognised by different combinations of CT-microsatellite and G–T SNP. Amylose content analysed in a random set of 39 genotypes was correlated with different microsatellite alleles/haplotypes. The highest Ac levels (>30%) correlated with (CT)10 and 16, high (26–30%) with (CT)11, 15 and 20, and intermediate (21–25%) with (CT)14, in all cases with G at the G–T SNP. The CT-classes (CT)17 and 18 (mean Ac value of 21%), could be subdivided into low amylose haplotypes (16–20%) for 17T and 18T and intermediate amylose haplotypes (21–25%) for 17G and 18G. The use of haplotypes proved to discriminate between intermediate and low amylose accessions within the same microsatellite class. Analyses of a segregating population of a cross between low and high Ac parents showed that CT-microsatellite may help to classify breeding lines and identify pollen contamination. We suggest that CT-microsatellite together with G–T SNP may be used as molecular marker by breeders to develop varieties with desired amylose levels.     

Polymorphisms in the ALS gene of weedy rice (Oryza sativa L.) accessions with differential tolerance to imazethapyr

Herbicide-resistant Clearfield™ rice technology allows the use of ALS inhibitors to control weedy rice. Weedy rice plants have differential tolerance to imazethapyr, perhaps due to ALS polymorphisms. We aimed to assess ALS polymorphisms in weedy rice accessions from Arkansas, USA, with differential tolerance to imazethapyr in seedling growth bioassays. Six base changes were identified in the ALS of 14 weedy rice accessions. Three of these nucleotide changes resulted in amino acid substitutions — Pro⁹³Thr, Glu⁶³⁰Asp, and Val⁶⁶⁹Met — in four accessions: Ark-4, Ark-9, Poi-1 and Poi-6. The GR₅₀ values and inhibition of root and shoot growth (%) of these accessions differed. The Glu⁶³⁰Asp substitution occurs in the herbicide binding domain B and Val⁶⁶⁹Met occurs at the C-terminal tail where the co-factor binds. Variability in weedy rice ALS exists, but polymorphism patterns did not relate to tolerance levels. The observed mutations presented the possibility that sustained selection pressure will eventually lead to selection of a herbicide-tolerant individual that will be the progenitor of a resistant population. Concomitantly, pollen-mediated gene flow from Clearfield™ rice to weedy rice will lead to the evolution of ALS-resistant weedy rice populations.     

Distribution of phytic acid and mineral elements in three indica rice (Oryza sativa L.) cultivars

Three indica rice cultivars with different grain shapes were selected to determine the milling characteristics and distribution of phytic acid (PA) and six mineral elements including magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), zinc (Zn), and selenium (Se). Milling characteristics were quite different among the rice cultivars with different physical dimensions. Similar milling times did not necessarily result in the same degree of milling (DOM) for different rice cultivars. The concentrations of phytic acid and minerals decreased with an increased DOM. These results also showed that phytic acid and minerals except for Zn and Se were not evenly distributed and highly concentrated in the outer layer (0% < DOM < 15%) of the rice kernel. In contrast, Zn and Se seem to be relatively evenly distributed in the grain. Optimum DOM of about 14% was detected for Zhenong 7 (long and slender grain); 10% was found for Zhenong 60 (medium-grain) and 9% for Zhenong 34 (short and round grain). The information generated in this experiment could be useful to optimize milling procedures for maximum removal of phytic acid, minimum mineral losses and weight loss in indica rice cultivars with different grain shapes.     

Genomic regions associated with the degree of red coloration in pericarp of rice (Oryza sativa L.)

The degree of red coloration (DRC) in pericarp of rice depends on the content of flavonoid compounds which have beneficial health effects for humans. In this study, 182 backcross-recombinant inbred lines (BILs) derived from Koshihikari (white pericarp)/Kasalath (red pericarp)//Koshihikari were used to detect the genomic regions associated with DRC through the QTL mapping approach. As a result, a total of four genomic regions were found to associate with DRC on chromosomes 1, 7, 9 and 11, respectively. Interestingly, the two genomic regions having the largest effects corresponded to previously characterized Rc and Rd genes on chromosome 7 and 1, respectively. In addition, two novel genomic regions having minor effects on DRC and located on chromosomes 9 and 11, respectively, are reported here for the first time. These results and the identification of tightly linked molecular markers that flank the genomic regions provide an opportunity for marker-aided improvement of red coloration in pericarp of rice.     

A set of near-isogenic lines for blast resistance genes with an Indica-type rainfed lowland elite rice (Oryza sativa L.) genetic background

A set of near-isogenic lines for blast resistance genes was developed by using an Indica-type elite rice variety, IR49830-7-1-2-2, suitable for the rainfed lowland conditions in the tropics, as a genetic background. Initially, we revealed that IR49830-7-1-2-2 harbors five blast resistance genes - Pia, Pib, Pik-s, Pita, and Pi11(t) - by using a differential system involving 19 selected standard blast isolates from the Philippines. Based on this result, we developed nine near-isogenic lines (NILs) targeting eight resistance genes - Pik, Pi7(t), Pi3, Pi5, Pita-2, Piz-5, Pish, and Pi9 - by recurrent backcrossing. The introgression of each resistance gene in the NILs was confirmed by reaction patterns to the blast isolates, allelism tests, and DNA marker analysis. In addition, a genome-wide DNA marker survey revealed that most of the chromosome regions in each NIL were of the IR49830-7-1-2-2 type. The agricultural characteristics of most of the developed NILs were almost the same as those of IR49830-7-1-2-2. Moreover, with one exception, they showed submergence tolerance similar to IR49830-7-1-2-2. The developed NILs could be used as a multiline variety suitable for the rainfed lowland in the tropics.     

Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran,embryo and endosperm of white,red and black rice kernels (Oryza sativa L.)

Whole rice grain comprising endosperm, embryo (or germ), and bran has potential beneficial health effects in addition to provision of nutrients. The distribution of phenolic acids and anthocyanins in endosperm, embryo, and bran of white, red, and black rice grains was investigated in this study. The total phenolic content (TPC) was highest in the bran, averaging 7.35 mg GAE/g and contributing 60%, 86% and 84% of phenolics in white, red and black rice. The average TPC of the embryo and endosperm were 2.79 and 0.11 mg GAE/g accounting for 17% and 23%, 4% and 10% and 7% and 9% in white, red and black rice, respectively. The antioxidant capacity determined using DPPH and ORAC displayed a trend similar to TPC. Free/conjugated phenolic acids in white, red and black rice bran accounted for 41%, 65% and 85% of total acids. Bound phenolic acids in rice bran accounted for 90% of total acids in whole grain. Cis-p-coumaric was detected in bound form in bran while cis-sinapic acid was detected in the free/conjugated form in embryo and bran. Cyanidin-3-O-glucoside and peonidin-3-O-glucoside were identified mainly in black rice bran as the total anthocyanins. Cyanindin-3-O-rutinoside was also detected in black rice bran.     

Yield potential and water use efficiency of aerobic rice (Oryza sativa L.) in Japan

Intensive rice farming in aerobic soil, referred to herein as aerobic rice, can greatly reduce the water input compared to that of flooded rice cultivation. The objective of this study was to compare the potential productivity of aerobic rice and flooded rice using high-yielding varieties at two locations in Japan in two successive years. In aerobic fields, the total amount of water supplied (irrigation plus rainfall) was 800–1300 mm. The soil water potential at 20-cm depth averaged between −15 and −30 kPa each growing season, but frequently reached −60 kPa. The average yield under aerobic conditions was similar to or even higher than that achieved with flooded conditions (7.9 t ha⁻¹ in 2007 and 9.4 t ha⁻¹ in 2008 for aerobic versus 8.2 t ha⁻¹ for flooded). The average water productivity under aerobic conditions was 0.8–1.0 kg grain m⁻³ water, slightly higher than common values in the literature. The super-high-yielding cultivar Takanari achieved yields greater than 10 t ha⁻¹ with no yield penalty under aerobic conditions in 3 out of 4 experiments. The favorable agronomic characteristic of Takanari was its ample sink capacity (grain number × grain weight). In conclusion, high-productivity rice cultivation in aerobic soil is a promising technology for water conservation. With continued breeding, future aerobic rice varieties will possess large numbers of spikelets and sufficient adaptation to aerobic conditions such that they will consistently achieve yields comparable to the potential yield of flooded rice.     

Effect of microwave irradiation on composition,structure and properties of rice (Oryza sativa L.) with different milling degrees

Effects of microwave irradiation on composition, structure, thermal and storage properties of rice with different milling degrees were investigated. Microwave irradiation partially inactivated the lipase and lipoxidase, damaged thiamine, and created puffed rice. In addition, the transition temperatures (T_o and T_P) of rice were shifted to higher temperatures, and the enthalpy of gelatinization decreased after irradiation. The effect was more obvious when the power of microwave was increased. Microstructure analysis showed that stress cracks and some “explosion” were created in rice kernels after microwave irradiation. Storage property analysis indicated that free fatty acid content of all rice samples increased gradually in the process of storage. Microwave irradiation reduced the release of free fatty acids during storage. The higher the power, the lesser the free fatty acids generated. The results suggested that microwave irradiation was potentially applicable for improving storage properties of brown and milled rice.