We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Higher plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidal non-mevalonate (MEP) pathway. Despite the compartmentalization of these two pathways, metabolic flow occurs between them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cross-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA insertion mutant lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, inhibitors of the MVA and MEP pathways, respectively. The accumulation of the major products of these pathways, i.e. sterols and chlorophyll, was less affected by lovastatin and clomazone, respectively, in loi1 than in the wild type. Furthermore, the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity analysis showed higher activity of HMGR in loi1-1 treated with lovastatin than that in the WT. We consider that the lovastatin-resistant phenotype of loi1-1 was derived from this post-transcriptional up-regulation of HMGR. The LOI1 gene encodes a novel pentatricopeptide repeat (PPR) protein. PPR proteins are thought to regulate the expression of genes encoded in organelle genomes by post-transcriptional regulation in mitochondria or plastids. Our results demonstrate that LOI1 is predicted to localize in mitochondria and has the ability to bind single-stranded nucleic acids. Our investigation revealed that the post-transcriptional regulation of mitochondrial RNA may be involved in isoprenoid biosynthesis in both the MVA and MEP pathways.

Investigations on concentration of mineral elements including Fe and Zn in wheat grains are important for human health. Two hundreds and sixtyfive cultivars and advanced lines were collected and sown at Anyang experimental station of the Institute of Crop Science of the Chinese Academy of Agriculture Sciences in season 2005-2006 to evaluate the genetic variation of major mineral element concentrations in wheat grain. Twenty-four selected cultivars were also planted at seven representative locations in seasons 2005-2006 and 2006-2007 to evaluate the effects of genotype, environment, and genotype by environment interaction on mineral element concentrations. The 265 genotypes displayed a large variation for all mineral elements investigated including Fe and Zn, ranging from 28.0 to 65.4 mg kg -1 and 21.4 to 58.2 mg kg -1 for Fe and Zn, with mean values of 39.2 and 32.3 mg kg -1 , respectively. Jimai 26, Henong 326, and Jingdong 8 displayed high Fe and Zn concentrations, and Jimai 26 and Henong 326 also displayed high concentrations of Cu, Mg, K, P, and protein content. Jingdong 8 is the most promising leading cultivar for increasing Fe and Zn concentrations. All mineral element concentrations including Fe and Zn were largely influenced by environment effects. Production of high Fe concentration can be best secured at Jiaozuo and Jinan, and high Zn concentration can be best secured at Jinan and Xuzhou, since samples from these locations in the two seasons are characterized by high Fe or Zn concentration, compared with the other locations. High and significant genotype by environment interaction effects on all mineral element concentrations were also observed, with ratios of genotype by environment to genotype variances all larger than 1.20. Grain Fe concentration was highly significant and positively correlated with that of Zn, indicating a high possibility to combine high Fe and Zn traits in wheat breeding. It also indicated strong positive correlations between concentrations of Fe, Zn, and protein content.

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Two hundred and seventy-three CIMMYT bread wheat cultivars and advanced lines grown under irrigated conditions in Mexico during the 2005-06 Yaqui crop cycle were characterized for qualityrelated genetic traits using gene-specific markers for some high-and low-molecular-weight glutenin subunit (HMW-GS and LMW-GS) genes, polyphenol oxidase (PPO), phytoene synthase (PSY), and waxy genes. Of them, 142 were analyzed for quality parameters including SDS sedimentation volume (SDS-SV), dough mixing time, and Alveograph parameters, and for HMW-GS and LMW-GS compositions using sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and reversedphase high-performance liquid chromatography (RP-HPLC). For the Ppo-A1 locus tested with the marker PPO18, the frequencies of alleles Ppo-A1a and PpoA1b were 79.1 and 20.2%, respectively, and no PCR fragment was amplified in 2 lines (0.73%), whereas 227 lines (83.2%) contained the allele Ppo-D1a and 46 lines (16.8%) had Ppo-D1b detected by markers PPO16 and PPO29. For the marker YP7A, 142 lines (52.0%) were assumed to have the allele Psy-A1a and 131 lines (48.0%) contained the allele Psy-A1b. In the case of the marker YP7B for the gene Psy-B1, the alleles Psy-B1a and Psy-B1b were detected in 155 (56.8%) and 43 (15.8%) lines, respectively, and 75 (27.4%) lines possessed the allele Psy-B1d detected by the marker YP7B-3. All 273 lines contained the alleles Wx-A1a and Wx-D1a as determined by markers MAG264 and MAG269, respectively. Using the marker Wx-B1, 204 lines (74.7%) were presumed to have the Wx-B1a allele and 69 (25.3%) possessed WxB1b. The over-expressing allele of Bx7 OE and subunit By8*, not clearly seen with SDS-PAGE, were detected by RP-HPLC. The numbers of lines with subunits Ax2*, By8, By9, Bx17, Bx20, Dx5, and Glu-B3j were 90, 16, 57, 5, 46, 118, and 33, respectively, in the 142 lines analyzed with molecular markers, and were consistent with the results obtained by SDS-PAGE, except for one line with the 1A.1R translocation. Subunits Ax1 and Ax2* at the Glu-A1 locus showed

SUMMARYUnlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

Four hundred and seventy-eight microsatellite markers derived from expressed sequence tags (EST-SSRs) were screened among three mapping populations (W-7984xOpata 85, WOpop; LumaixHanxuan, LHpop; WenmaixShanhongmai, WSpop). The number of polymorphic EST-SSR primer pairs found in WOpop, LHpop and WSpop was 92, 58 and 29 respectively. A total of 101 EST-SSR loci amplified from 88 primer sets were distributed over the 20 chromosomes of the reference maps (no markers were located on chromosome 4B). These 101 mapped EST-SSR markers add to the existing 450 microsatellite loci previously mapped in bread wheat. Seventy-four of the 101 loci showed significant similarities to known genes, including 24 genes involved in metabolism, 4 in cellular structures, 9 in stress resistance, 12 in transcription, 2 in development, 2 transporters and 21 storage proteins. Besides gliadin and glutenin, most of the 53 genes with putative functions were mapped for the first time by EST-SSR markers in bread wheat. Sequence alignment of the mapped wheat EST-SSR loci allowed tentative assignment of functionality to the other members of grasses family. Colinearity combined with homology information offers an attractive approach to comparative genomics.

One of the key targets of breeding programs in bread wheat is to improve the end-use quality. The relationships between quantities of protein fractions and dough rheological characters have been well established, but there is little information on the genetic control of quantities of protein fractions. Two hundred and forty F(6) recombinant inbred lines derived from a cross between two Chinese wheat cultivars, PH82-2 and Neixiang 188, were sown at Jiaozuo in Henan province in the 2005-2006 and 2006-2007 cropping seasons, and inclusive composite interval mapping was used to dissect main effect quantitative trait loci (M-QTLs) and digenic epistatic QTLs (E-QTLs) for quantities of protein fractions. A total of 55 M-QTLs and 77 pairs of E-QTLs affecting the quantities of protein fractions including GLU-A1 (QGA1), GLU-B1 (QGB1), GLU-D1 (QGD1), HMW-GS (QHMW), GLU-A3 (QGA3), GLU-B3 (QGB3), LMW-GS (QLMW), glutenin (QGLU) and the ratio of the quantity of glutenin to those of gliadin were identified, with M-QTLs contributing 39.3-95.6% of the phenotypic variance explained (PVE), and E-QTLs accounting for 1.4-33.5% of the PVE. Among the M-QTLs, 33 were consistent in two seasons and in the mean value of two seasons with similar effects in both magnitude and direction, including major genes on HMW and LMW glutenin loci linked to Sec1 and Glu-B1c, Glu-D1d, Glu-A3a, and grain hardness locus Ha, indicating that these genes were the most important determinants of gluten strength, and they might have significant effects on dough properties not only through effects on allelic composition, but also by influencing quantities of protein fractions. The effects of E-QTLs were more influenced by environments, compared with those of M-QTLs, with only two pairs of E-QTLs consistent in two seasons and in the mean value of two seasons. The M-QTLs were detected in 12 marker intervals, all of which involved E-QTLs on quantities of protein fractions, whereas only 40 of 77 pairs of E-QTLs involved intervals in which M-QTLs were detected. The results indicated that besides main effects, epistatic effects were also important factors in determining quantities of protein fractions in wheat.