Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity

When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.     

CRUMPLED LEAF (CRL) homologs of Physcomitrella patens are involved in the complete separation of dividing plastids

Plastid division is controlled by numerous nuclear genes. Arabidopsis thaliana CRUMPLED LEAF (AtCRL) is a plastid division-related gene, and the crl mutant exhibits a dwarf phenotype with abnormal cell division and a significant reduction in plastid numbers. However, the function of AtCRL is not fully understood. Here, we identified and characterized two AtCRL homologs, PpCRL1 and PpCRL2, in the moss Physcomitrella patens. PpCRL1 and PpCRL2 shared 77% amino acid identity with each other and 47% identity with AtCRL. Single PpCRL1 or -2 gene knockout (KO) mutants could not be distinguished from the wild-type mosses, but PpCRL1 and -2 double KO mutants displayed growth retardation of protonemata and gametophores and harbored approximately 10 large chloroplasts per cell. This indicates that PpCRL1 and PpCRL2 have redundant functions in chloroplast division and plant growth. Unlike the A. thaliana crl mutants, however, the PpCRL double KO mutants did not display abnormal orientation of the cell division plane. Complementation experiments showed that AtCRL partially rescued the defects in chloroplast size and number of the PpCRL double KO mutant. This suggests that PpCRL has a similar, but not identical, function to AtCRL. Time-lapse microscopic observation of the double PpCRL KO mutants revealed that some dumbbell-shaped chloroplasts failed to complete division at the late stage of plastid division; enlarged chloroplasts were thus generated. This strongly suggests that PpCRLs are involved in the complete separation of dividing chloroplasts.     

Production of bioactive compounds based on phylogeny in the genus Penicillium preserved at NBRC

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.     

Usefulness of sugar mapping by liquid chromatography/mass spectrometry in comparability assessments of glycoprotein products.

We have previously reported that sugar-mapping by liquid chromatography/mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) can be useful for structural analysis of carbohydrates in a glycoprotein. In this paper, we evaluated sugar-mapping with regard to its use in comparability assessment of glycoprotein products. Erythropoietins (EPO) produced from three different sources were chosen as models of the closely related glycoprotein products. The two-dimensional displays of sugar maps drawn by LC/MS with GCC clearly showed the differences in carbohydrate heterogeneity with regard to sialylation, acetylation, and sulphation patterns among three EPOs. Exoglycosidase digestion followed by sugar-mapping provided information regarding the structure of characteristic carbohydrates in each EPO. These results demonstrate that LC/MS with GCC can reveal the details of carbohydrate heterogeneity in order to distinguish between closely related glycoprotein products. Our method can thus be useful in comparability assessments of therapeutic glycoproteins.     

Micro determination of unsaturated disaccharide formed by the action of acidic glycosaminoglycan-endoeliminases. An application of the thiobarbituric acid method to the assay of D-gluco-4-enepyranosyluronic acid-containing disaccharides.

An improved method is described for the micro determination of acidic glycosaminoglycans after digestion with chondroitinase-ABC and -AC. The determination is based on the color production of D-gluco-4-enepyranosyluronic acid-containing disaccharides produced by the action of chondroitinase-ABC and -AC (acidic glycosaminoglycans-endoeliminase) when the periodate-thiobarbituric acid method is applied to the alpha,beta-unsaturated disaccharides. Suitable conditions for the quantitative assay are described.     

Strain-Dependent Differences in Locomotor Activity after Local Brain Irradiation with 30 GyE of Carbon Ions.

This study investigated strain differences in brain damage among male A/J, C57BL/6JNrs and C3H/HeNrs mice after local brain irradiation. Whole brains were irradiated with a single dose of 30 GyE carbon ion beams and then locomotor activity was determined as body heat of each animal. The daily locomotor activities of untreated mice differed among strains. Non-irradiated C57BL/6JNrs mice were more active than A/J mice. This variance became more obvious immediately after irradiation, when the activity of A/J and C3H/HeNrs mice diminished, whereas that of C57BL/6JNrs mice increased at the beginning of the active phase and remained elevated for three days after irradiation. The altered activities of all three strains of irradiated mice gradually recovered to normal within three to four days.     

Prion inactivation by the Maillard reaction

Since variant Creutzfeldt-Jakob disease (vCJD) has been suspected to be attributable to the infectious agents associated with bovine spongiform encephalopathy (BSE), it is important to prevent the transmission of pathogenic forms of prion protein (PrP(Sc)) through contaminated feeding materials such as meat and bone meal (MBM). Here, we demonstrate that the Maillard reaction employing a formulation of glucose in combination with sodium hydrogen carbonates effectively reduced the infectivity (approximately 5.9-log reduction) of a scrapie-infected hamster brain homogenate. In addition to a bioassay, a protein misfolding cyclic amplification (PMCA) technique, in which PrP(Sc) can be amplified in vitro, was used as a rapid test for assessing PrP(Sc) inactivation. The PMCA analysis also indicated that the PrP(Sc) level in the infected material significantly decreased following the Maillard reaction. Therefore, the Maillard reaction can be employed for the decontamination of large amounts of byproducts such as MBM.