Fine mapping and candidate gene analysis of LM3, a novel lesion mimic gene in rice

A rice lesion mimic mutant, lm3, was obtained by the mutagenesis of an indica cultivar, 93-11, using γ-ray radiation. Brownish lesions appeared on the leaves of lm3 at the young seedling stage and persisted until the ripening stage. The lm3 mutant was characterised by a shorter plant height and delayed heading compared with the wild-type 93-11. A genetic analysis indicated that the lesion mimic phenotype was controlled by a single recessive gene. Using simple sequence repeat (SSR) markers, the target gene LM3 was first located between marker RM5748 and RM14906 on chromosome 3. We then developed Insertion-Deletion (InDel) markers to fine-map LM3, and the locus was localised to a 29 kb region defined by two InDel markers, In12571 and In12600. Five ORFs were predicted in the candidate region, and DNA sequencing detected a single-nucleotide polymorphism (SNP) in the coding region of LOC Os03g21900. The SNP in the fourth exon (C in 93-11; T in lm3) of LOC_Os03g21900 results in the substitution of a proline (P) with a serine (S) at the 140^th amino acid of the deduced uroporphyrinogen decarboxylase protein. We did not detect polymorphisms in the other predicted ORF regions between lm3 and 93-11. These results suggest that LOC_Os03g21900 is the most likely candidate gene for LM3.     

Determination of the genetic architecture of seed size and shape via linkage and association analysis in soybean (Glycine max L. Merr.)

Seed-size traits, which are controlled by multiple genes in soybean, play an important role in determining seed yield, quality and appearance. However, the molecular mechanisms controlling the size of soybean seeds remain unclear, and little research has been done to investigate these mechanisms. In this study, we performed a genetic analysis to determine the genetic architecture of soybean seed size and shape via linkage and association analyses. We used 184 recombinant inbred lines (RILs) and 219 cultivated soybean accessions to evaluate seed length, seed width and seed height as seed-size traits, and their ratios of these values as seed-shape traits. Our results showed that all six traits had high heritability ranging from 92.46 to 98.47 %. Linkage analysis in the RILs identified 12 quantitative traits loci (QTLs), with five of these QTLs being associated with seed size, five with seed shape and two with the two first principal components of our principal component analysis (PCA). Association analysis in the 219 accessions detected 41 single nucleotide polymorphism (SNP)-trait associations, with 20 of these SNPs being associated with seed-size traits, seven with seed-shape traits and 14 with the two first principal components of our PCA. This analysis reveals that seed-size and seed-shape may be controlled by different genetic factors. Our results provide a greater understanding of phenotypic structure and genetic architecture of soybean seed, and the QTLs detected in this study form a basis for future fine mapping, quantitative trait gene cloning and molecular breeding in soybean.     

ZmGA3ox2, a candidate gene for a major QTL,qPH3.1, for plant height in maize

Maize plant height is closely associated with biomass, lodging resistance and grain yield. Determining the genetic basis of plant height by characterizing and cloning plant height genes will guide the genetic improvement of crops. In this study, a quantitative trait locus (QTL) for plant height, qPH3.1, was identified on chromosome 3 using populations derived from a cross between Zong3 and its chromosome segment substitution line, SL15. The plant height of the two lines was obviously different, and application of exogenous gibberellin A₃ removed this difference. QTL mapping placed qPH3.1 within a 4.0 cM interval, explaining 32.3% of the phenotypic variance. Furthermore, eight homozygous segmental isolines (SILs) developed from two larger F₂ populations further narrowed down qPH3.1 to within a 12.6 kb interval. ZmGA3ox2, an ortholog of OsGA3ox2, which encodes a GA3 β‐hydroxylase, was positionally cloned. Association mapping identified two polymorphisms in ZmGA3ox2 that were significantly associated with plant height across two experiments. Quantitative RT‐PCR showed that SL15 had higher ZmGA3ox2 expression relative to Zong3. The resultant higher GA₁ accumulation led to longer internodes in SL15 because of increased cell lengths. Moreover, a large deletion in the coding region of ZmGA3ox2 is responsible for the dwarf mutant d1‐6016. The successfully isolated qPH3.1 enriches our knowledge on the genetic basis of plant height in maize, and provides an opportunity for improvement of plant architecture in maize breeding.     

Sterols are required for cell‐fate commitment and maintenance of the stomatal lineage in Arabidopsis

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk–J3158) of the Arabidopsis sterol C–14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell‐fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk–J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α–demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C–14 reductase in Arabidopsis, also caused these abnormal small‐cell and stomata phenotypes in wild‐type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time‐lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell‐fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.     

A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [³H]5-HT uptake in COS-7 cells (K_i = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative to the transporters for dopamine and norepinephrine. Visualization of the SERT with compound 8 was demonstrated by confocal microscopy in HEK293 cells stably expressing EGFP–SERT.     

Synthesis and evaluation of carbamoylmethylene linked prodrugs of BMS-582949, a clinical p38α inhibitor

A series of carbamoylmethylene linked prodrugs of 1 (BMS-582949), a clinical p38α inhibitor, were synthesized and evaluated. Though the phosphoryloxymethylene carbamates (3, 4, and 5) and α-aminoacyloxymethylene carbamates (22, 23, and 26) were found unstable at neutral pH values, fumaric acid derived acyloxymethylene carbamates (2, 28, and 31) were highly stable under both acidic and neutral conditions. Prodrugs 2 and 31 were also highly soluble at both acidic and neutral pH values. At a solution dose of 14.2 mpk (equivalent to 10 mpk of 1), 2 gave essentially the same exposure of 1 compared to dosing 10 mpk of 1 itself. At a suspension dose of 142 mpk (equivalent to 100 mpk of 1), 2 demonstrated that it could overcome the solubility issue associated with 1 and provide a much higher exposure of 1. To our knowledge, the unique type of prodrugs like 2, 28, and 31 was not reported in the past and could represent a novel prodrug approach for secondary amides, a class of molecules frequently identified as drug candidates.     

4-Bicyclic heteroaryl-piperidine derivatives as potent,orally bioavailable Stearoyl-CoA desaturase-1 (SCD1) inhibitors. Part 1: Urea-based analogs

A new series of urea-based, 4-bicyclic heteroaryl-piperidine derivatives as potent SCD1 inhibitors is described. The structure–activity relationships focused on bicyclic heteroarenes and aminothiazole–urea portions are discussed. A trend of dose-dependent decrease in body weight gain in diet-induced obese (DIO) mice is also demonstrated.