BnaC7.ROT3, the causal gene of cqSL-C7, mediates silique length by affecting cell elongation in Brassica napus

Siliques are a major carbohydrate source of energy for later seed development in rapeseed (Brassica napus). Thus, silique length (SL) has received great attention from breeders. We previously detected a novel quantitative trait locus cqSL-C7 that controls SL in B. napus. Here, we further validated the cqSL-C7 locus and isolated its causal gene (BnaC7.ROT3) by map-based cloning. In Zhongshuang11 (parent line with long siliques), BnaC7.ROT3 encodes the potential cytochrome P450 monooxygenase CYP90C1, whereas in G120 (parent line with short siliques), a single nucleotide deletion in the fifth exon of BnaC7.ROT3 results in a loss-of-function truncated protein. Subcellular localization and expression pattern analysis revealed that BnaC7.ROT3 is a membrane-localized protein mainly expressed in leaves, flowers and siliques. Cytological observation showed that the cells in silique wall of BnaC7.ROT3-transformed positive plants were longer than those of transgene-negative plants in the background of G120, suggesting that BnaC7.ROT3 affects cell elongation. Haplotype analysis demonstrated that most of the alleles of BnaC7.ROT3 are favorable alleles in B. napus germplasms and its homologs may also be involved in SL regulation. Our findings provide novel insights into the regulatory mechanisms of natural SL variations and valuable genetic resources for the improvement of SL in B. napus.

Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells

Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase. Disclosures: No relevant conflicts of interest to declare.

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged α- or β-subunits of the epithelial Na+channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the α- and β-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+transport.

Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L.)

With 18 strains of Rhizobium leguminosarum bv. phaseoli the bean mutants R99 and NOD125 remained essentially non-nodulating, while the mutant R69 produced a variable number of small white ineffective nodules, and the wild-type parent-line OAC Rico formed a variable number of pink effective nodules. Both R69 and R99 grew less vigorously than OAC Rico, but possessed similar levels of nitrate reductase in both roots and leaves, and responded in a normal way to increased supply of combined nitrogen. Reciprocal grafts between the non-nodulating R99 and NOD125, the ineffective R69, the wild-type parent line OAC Rico, and the supernodulating R32BS, demonstrated that the non-nodulating and ineffective characters were controlled by the root, and confirmed that the supernodulation character was controlled by the shoot. Key words: Common bean, nitrate reductase, non-fixing mutants, Phaseolus vulgaris, Rhizobium strains, supernodulation

EFFECTS OF Rhizobium INOCULUM CONCENTRATION STRAIN AND COMBINED NITROGEN ON GROWTH AND NODULATION OF A SUPERNODULATING COMMON BEAN AND ITS PARENT LINE

Numbers of nodules formed on both the supernodulating common bean (Phaseolus vulgaris L.) line R32-BS (BS) and its parent cultivar (OAC Rico) increased with Rhizobium inoculum concentration up to about 105 cells mL−1. As numbers of nodules increased, their average size decreased. Red nodules as a fraction of total nodules decreased with increasing numbers of nodules: a large proportion of nodules at the higher concentrations appeared to be non-functional. Acetylene reduction (AR) rates per plant did not differ between supernodulator and parent line. AR per gram total nodule fresh weight was greater in the parent line than in the supernodulator, but AR per gram red nodule weight was the same in the two lines. Combined nitrogen had its main effect in increasing plant weight, but had little effect on nodule characteristics. However, nodule weights/numbers per unit plant weight were decreased by combined nitrogen. OAC Rico and BS responded similarly to N. Rhizobium strain TAL182 produced fewer but more efficient nodules than Kim5s.Key words: Rhizobium leguminosarum bv. phaseoli, Phaseolus vulgaris, supernodulation, nodule number, nodule senescence, inoculum concentration