Characterization and expression analyses of two plastidic enolase genes in rice

To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, OsEno1 and OsEno3 encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::GUS analysis showed that OsEno3 was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of OsEno1 was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase in vivo, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts.     

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.     

Glutelin subtype-dependent protein localization in rice grain evidenced by immunodetection analyses

Plant Molecular Biology - GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to...     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

Quantitative trait locus analyses of ozone-induced grain yield reduction in rice

Reduction of grain yield (total seed weight) by ozone in rice (Oryza sativa L.) is believed to be caused by ozone-induced reduction of photosynthetic activity followed by growth inhibition. Here, japonica rice cultivar Sasanishiki and indica rice cultivar Habataki showed different responses to ozone. When exposed to ozone, the leaves of Habataki exhibited no critical damage, whereas those of Sasanishiki developed lesions. Conversely, ozone exposure reduced total seed weight by 19% in Habataki, but not significantly in Sasanishiki. Chronic ozone exposure also significantly decreased culm length, number of primary rachis branch, and number of spikelets per panicle in Habataki. QTL analysis in Sasanishiki/Habataki chromosome segment substitution lines identified a single locus associated with the yield loss caused by ozone on chromosome 6 of Habataki close to marker RM3430 (107.6 cM). A QTL for reduction of primary rachis branch number and total spikelet number was found in the same position. These results indicate that a QTL on chromosome 6 has an important role in ozone-induced yield loss, and is also involved in primary rachis branch formation and total spikelet number in ozone-exposed rice.     

Succession of methanogenic archaea in rice straw incorporated into a Japanese rice field: estimation by PCR-DGGE and sequence analyses

The succession and phylogenetic profiles of methanogenic archaeal communities associated with rice straw decomposition in rice-field soil were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by 16S rDNA sequencing. Nylon bags containing either leaf sheaths or blades were buried in the plowed layer of a Japanese rice field under drained conditions during the off-crop season and under flooded conditions after transplanting. In addition, rice straw samples that had been buried in the rice field under drained conditions during the off-crop season were temporarily removed during spring plowing and then re-buried in the same rice field under flooded conditions at transplanting. Populations of methanogenic archaea were examined by amplification of the 16S rRNA genes in the DNA extracted from the rice straw samples. No PCR product was produced for samples of leaf sheath or blade prior to burial or after burial under drained conditions, indicating that the methanogen population was very small during decomposition of rice straw under oxic conditions. Many common bands were observed in rice straw samples of leaf sheath and blade during decomposition of rice straw under flooded conditions. Cluster analysis based on DGGE patterns divided methanogenic archaeal communities into two groups before and after the mid-season drainage. Sequence analysis of DGGE bands that were commonly present were closely related to Methanomicrobiales and Rice cluster I. Methanomicrobiales, Rice cluster I and Methanosarcinales were major members before the mid-season drainage, whereas the DGGE bands that characterized methanogenic archaeal communities after the mid-season drainage were closely related to Methanomicrobiales. These results indicate that mid-season drainage affected the methanogenic archaeal communities irrespective of their location on rice straw (sheath and blade) and the previous history of decomposition during the off-crop season.     

Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.     

Metabolomic and functional genomic analyses reveal varietal differences in bioactive compounds of cooked rice

Emerging evidence supports that cooked rice (Oryza sativa L.) contains metabolites with biomedical activities, yet little is known about the genetic diversity that is responsible for metabolite variation and differences in health traits. Metabolites from ten diverse varieties of cooked rice were detected using ultra performance liquid chromatography coupled to mass spectrometry. A total of 3,097 compounds were detected, of which 25% differed among the ten varieties. Multivariate analyses of the metabolite profiles showed that the chemical diversity among the varieties cluster according to their defined subspecies classifications: indica, japonica, and aus. Metabolite-specific genetic diversity in rice was investigated by analyzing a collection of single nucleotide polymorphisms (SNPs) in genes from biochemical pathways of nutritional importance. Two classes of bioactive compounds, phenolics and vitamin E, contained nonsynonymous SNPs and SNPs in the 5' and 3' untranslated regions for genes in their biosynthesis pathways. Total phenolics and tocopherol concentrations were determined to examine the effect of the genetic diversity among the ten varieties. Per gram of cooked rice, total phenolics ranged from 113.7 to 392.6 μg (gallic acid equivalents), and total tocopherols ranged between 7.2 and 20.9 μg. The variation in the cooked rice metabolome and quantities of bioactive components supports that the SNP-based genetic diversity influenced nutritional components in rice, and that this approach may guide rice improvement strategies for plant and human health.     

Inheritance of early elongation ability in floating rice revealed by diallel and QTL analyses

In floating rice, stem elongation begins much earlier than in non-floating rice, which is the major survival mechanism for flooding. Inheritance of this early elongation ability was studied using diallel and quantitative trait locus (QTL) analyses. The diallel analysis was undertaken using a set of 6×6 half-diallel crosses involving four floating (Goai, Habiganj Aman VIII, Badal 106, and Oryza rufipogon strain W120) and two non-floating (Latisail and Patnai 23) parents. The additive gene effects were higher than the dominant effects. The dominant alleles were concentrated in the cultivated floating parents (Goai, Habiganj Aman VIII, and Badal 106), whereas the recessive alleles were in the wild floating parent (W120). A QTL analysis using a Patnai 23 × Goai F₂ population detected two putative QTLs. Of these QTLs, the one on chromosome 12 behaved as a partially dominant major gene that explained more than half of the total genetic variation.Communicated by D.J. Mackill     

Molecular and functional analyses of COPT/Ctr-type copper transporter-like gene family in rice

Background  

The copper (Cu) transporter (COPT/Ctr) gene family has an important role in the maintenance of Cu homeostasis in different species. The rice COPT-type gene family consists of seven members (COPT1 to COPT7). However, only two, COPT1 and COPT5, have been characterized for their functions in Cu transport.