Signaling pathway perturbation analysis for assessment of biological impact of cigarette smoke on lung cells.

Exposure to cigarette smoke (CS) results in injury to the epithelial cells of the human respiratory tract and has been implicated as a causative factor in the development of chronic obstructive pulmonary disease and lung cancers. The application of omics-scale methodologies has improved the capacity to understand cellular signaling processes underlying response to CS exposure. We report here the development of an algorithm based on quantitative assessment of transcriptomic profiles and signaling pathway perturbation analysis (SPPA) of human bronchial epithelial cells (HBEC) exposed to the toxic components present in CS. HBEC were exposed to CS of different compositions and for different durations using an ISO3308 smoking regime and the impact of exposure was monitored in 2263 signaling pathways in the cell to generate a total effect score that reflects the quantitative degree of impact of external stimuli on the cells. These findings support the conclusion that the SPPA algorithm provides an objective, systematic, sensitive means to evaluate the biological impact of exposures to CS of different compositions making a powerful comparative tool for commercial product evaluation and potentially for other known or potentially toxic environmental smoke substances.

Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

Quantitative trait loci analysis of brown blotch resistance in cowpea variety KN1.

Cowpea is one of the most important crops in West Africa and is essential for the region's food and nutrition security and economic development. Consequently, improving its agronomic performance and yield is a desirable goal. Brown blotch disease, caused by the fungal pathogen Colletotrichum capsici, is an important constraint of cowpea productivity, and at present, only limited genetic resources are available for breeding improved brown blotch-resistant varieties. The current study has characterized the genetic basis for brown blotch resistance conferred by the cowpea cultivar KN1 and identified a major dominant quantitative trait locus (QTL) for resistance on chromosome Vu02. A segregating F2 population (n = 200), derived from a cross between KN1 and brown blotch-susceptible Tiligre (KVx775-33-2G), was developed and scored for disease severity following controlled inoculation. A subset of the population (n = 94) was genotyped with 99 newly developed allele-specific polymerase chain reaction (AS-PCR) markers, and multiple interval mapping was performed. One major and three minor QTL were identified. This is the first reported mapping of QTL conferring resistance to C. capsici in cowpea, and it is expected that the markers identified here will be a valuable resource for developing elite cowpea cultivars with resistance to brown blotch.