Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Are rice chromosomes components of a holocentric chromosome ancestor?

Comparative genomics reveals that cereal genomes are composed of similar genomic building blocks (linkage blocks). By stacking these blocks in a unique order, it is possible to construct a single ancestral chromosome which can be cleaved to give the basic structure of the 56 different chromosomes found in wheat, rice, maize, sorghum, millet and sugarcane. The borders of linkage blocks are defined by cereal centromeric and telomeric sites. However, a number of studies have shown that telomeric heterochromatin has neocentromeric activity, implying that linkage blocks are in fact defined by centromeric-like sites with conserved sequences. The structure of the ancestral cereal genome thus resembles a holocentric chromosome, which is the chromosome structure shared by the closest relatives of the Gramineae, the Cypericeae and Juncaceae.     

Effects of magnesium deficiency on magnesium uptake activity of rice root,evaluated using <Superscript>28</Superscript> Mg as a tracer

Aims

The mechanisms underlying magnesium (Mg) uptake by plant roots remain to be fully elucidated. In particular, there is little information about the effects of Mg deficiency on Mg uptake activity. A Mg uptake kinetic study is essential for better understanding the Mg uptake system.

Methods

We performed a Mg uptake tracer experiment in rice plants using ²⁸?Mg.

Results

Mg uptake was mediated by high- and low-affinity transport systems. The K _m value of the high-affinity transport system was approximately 70 μM under Mg-deficient conditions. The Mg uptake activity was promoted by Mg deficiency, which in turn fell to the basal level after 5- min of Mg resupply. The induced uptake rate was inhibited by ionophore treatment, suggesting that an energy-dependent uptake system is enhanced by Mg deficiency.

Conclusions

The Mg uptake changes rapidly with Mg conditions in rice, as revealed by a ²⁸?Mg tracer experiment. This technique is expected to be applicable for Mg uptake analyses, particularly in mutants or other lines.

     

Characterization of the putative α subunit of a heterotrimeric G protein in rice

A recombinant protein with a cDNA that encodes the putative subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice ) with an apparent molecular mass of 45 kDa was bound with guanosine 5-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice . A 45 kDa polypeptide in the plasma membrane, as well as rGrice , was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the subunits of heterotrimeric G proteins in animals, suggesting that the putative subunit is truly the subunit itself.     

Does Bt rice pose risks to non‐target arthropods? Results of a meta‐analysis in China

Transgenic Bt rice expressing the insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) has been developed since 1989. Their ecological risks towards non‐target organisms have been investigated; however, these studies were conducted individually, yielding uncertainty regarding potential agroecological risks associated with large‐scale deployment of Bt rice lines. Here, we developed a meta‐analysis of the existing literature to synthesize current knowledge of the impacts of Bt rice on functional arthropod guilds, including herbivores, predators, parasitoids and detritivores in laboratory and field studies. Laboratory results indicate Bt rice did not influence survival rate and developmental duration of herbivores, although exposure to Bt rice led to reduced egg laying, which correctly predicted their reduced abundance in Bt rice agroecosystems. Similarly, consuming prey exposed to Bt protein did not influence survival, development or fecundity of predators, indicating constant abundances of predators in Bt rice fields. Compared to control agroecosystems, parasitoid populations decreased slightly in Bt rice cropping systems, while detritivores increased. We draw two inferences. One, laboratory studies of Bt rice showing effects on ecological functional groups are mainly either consistent with or more conservative than results of field studies, and two, Bt rice will pose negligible risks to the non‐target functional guilds in future large‐scale Bt rice agroecosystems in China.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Responses of a rice–wheat rotation agroecosystem to experimental warming

Climate change is likely to affect agroecosystems in many ways. This study was performed to investigate how a rice–winter wheat rotation agroecosystem in southeast China would respond to global warming. By using an infrared heater system, the soil surface temperature was maintained about 1.5 °C above ambient milieu over 3 years. In the third growing season (2009–2010), the evapotranspiration (ET) rate, crop production, soil respiration, and soil carbon pool were monitored. The ET rate was 23 % higher in the warmed plot as compared to the control plot during the rice paddy growing season, and the rice grain yield was 16.3 % lower, but there was no significant difference in these parameters between the plots during the winter wheat-growing season. The phenology of the winter wheat shifted under experimental warming, and ET may decrease late in the winter wheat-growing season. Experimental warming significantly enhanced soil respiration, with mean annual soil respiration rates of 2.57 ± 0.17 and 1.96 ± 0.06 μmol CO₂ m^?2 s^?1 observed in the warmed and control plots, respectively. After 3 years of warming, a significant decrease in the total organic carbon was observed, but only in the surface soil (0–5 cm). Warming also stimulated the belowground biomass, which may have compensated for any heat-induced loss of soil organic carbon. Paddy rice seemed to be more vulnerable to warming than winter wheat in terms of water-use efficiency and grain production.     

Genetic analysis of genetic basis of a physiological disorder “straighthead” in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Straighthead is a physiological disorder in rice that causes yield losses and is a serious threat to rice production worldwide. Identification of QTL conferring resistance will help develop resistant cultivars for straighthead control. We conducted linkage mapping to identify QTL involved with straighthead. The study was based on a F₂ population developed from a cross between ‘Zhe733(resistant)/R312(susceptible)’. Using phenotypic data of F₂ plants and their F_2:3 families, two major QTL, qSTH-2 and qSTH-8, were identified using bulked segregant analysis, explaining 11.1 and 28.1 % of the phenotypic variation on chromosome 2 and 8, respectively. The qSTH-2 for straighthead resistance was identified by linkage mapping. qSTH-2 was situated near a QTL “AsS” responsible for arsenic accumulation. Straighthead is frequently observed on land where As has accumulated. The result suggests a kind of internal connection between qSTH-2 and AsS. Additionally, the QTL qSTH-8 was located close to HD5 related with heading date. The close location may be associated with the observation of early heading among straighthead resistant varieties. These findings should be useful for further genetic study of straighthead.     

Selection of efficient vesicular-arbuscular mycorrhizal fungi for wetland rice — a preliminary screen

Eighteen different VA mycorrhizal fungi were screened for their symbiotic responses with wet land rice cultivar Prakash (IET 2254) under pot culture. Of the 18 fungi, Acaulospora sp. (ICRISAT), Glomus fasciculatum (Riverside) and G. mosseae (Invermay) were found to be the best in improving plant height, tiller number, total biomass, panicle number, grain weight and plant P and Zn concentrations. Increases in grain yield caused by inoculation with Acaulospora sp. (ICRISAT), G. fasciculatum (Riverside) and G. mosseae (Invermay) were 62%, 59% and 35%, respectively.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Natural alleles of a uridine 5ʹ-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5ʹ-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1^YZN compared to EDR1^YD1, resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1^YD1 and EDR1^YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1^YD1 and EDR1^YZN varieties indicated that rice varieties harboring EDR1^YZN and EDR1^YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.     

Molecular cloning and expression characterization of a rice K+ channel β subunit

K⁺ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming subunit and the subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K⁺ channel subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K⁺ channel subunits, and 72% identity with the only other cloned plant (Arabidopsis thaliana) K⁺ channel subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K⁺ channel subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K⁺ channel subunit.Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K⁺ and act as reserve sources of this cation when new growth requires remobilization of K⁺. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K⁺ was lost from older leaves of plants grown on K⁺-deficient fertilizer, the loss of cellular K⁺ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K⁺ status may specifically alter expression of a gene encoding a K⁺ channel subunit.     

Sequence analysis of the long arm of rice chromosome 11 for rice–wheat synteny

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10× redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.