Reducing Crop Production Cost

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Most Asked Agronomic Questions

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A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms

BACKGROUND: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. RESULTS: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. CONCLUSION: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes

Structural and micro-anatomical changes in vertebrae associated with idiopathic-type spinal curvature in the curveback guppy model

Background: The curveback lineage of guppy is characterized by heritable idiopathic-type spinal curvature thatdevelops during growth. Prior work has revealed several important developmental similarities to the human idiopathicscoliosis (IS) syndrome. In this study we investigate structural and histological aspects of the vertebrae that areassociated with spinal curvature in the curveback guppy and test for sexual dimorphism that might explain a femalebias for severe curve magnitudes in the population.Methods: Vertebrae were studied from whole-mount skeletal specimens of curved and non-curved adult males andfemales. A series of ratios were used to characterize structural aspects of each vertebra. A three-way analysis of variancetested for effects of sex, curvature, vertebral position along the spine, and all 2-way interactions (i.e., sex and curvature,sex and vertebra position, and vertebra position and curvature). Histological analyses were used to characterize microarchitecturalchanges in affected vertebrae and the intervertebral region.Results: In curveback, vertebrae that are associated with curvature demonstrate asymmetric shape distortion,migration of the intervertebral ligament, and vertebral thickening on the concave side of curvature. There is sexualdimorphism among curved individuals such that for several vertebrae, females have more slender vertebrae than domales. Also, in the region of the spine where lordosis typically occurs, curved and non-curved females have a reducedwidth at the middle of their vertebrae, relative to males.Conclusions: Based on similarities to human spinal curvatures and to animals with induced curves, the concaveconvexbiases described in the guppy suggest that there is a mechanical component to curve pathogenesis incurveback. Because idiopathic-type curvature in curveback is primarily a sagittal deformity, it is structurally more similarto Scheuermann kyphosis than IS. Anatomical differences between teleosts and humans make direct biomechanicalcomparisons difficult. However, study of basic biological systems involved in idiopathic-type spinal curvature incurveback may provide insight into the relationship between a predisposing aetiology, growth, and biomechanics.Further work is needed to clarify whether observed sex differences in vertebral characteristics are related to the femalebias for severe curves that is observed in the population

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB

Cyclin-Dependent Kinase 6 Phosphorylates NF-kappa B P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression

Peer reviewe

Dissolution Behavior of Ternary Alkali-Alkaline Earth-Borate Glasses in Water

The compositional dependence of the dissolution rates of ternary alkali-alkaline earth borate glasses in water were characterized. These glasses dissolve congruently and the dissolution kinetics were linear, following the contracting volume model for spherical particles. The dissolution rates were dependent on the glass structure, with slower rates associated with glasses with greater fractions of four-coordinated borate sites in their network structures, as determined by nuclear magnetic resonance spectroscopy. For glasses with a fixed alkaline earth identity, the dissolution rates increased in the order Li \u3c Na \u3c K. For glasses with a constant alkali identity, the dissolution rates increased in the order Ca \u3c Sr ≈ Mg. The effects of composition on dissolution rates are discussed in terms of the reactions between water and different borate and metal-oxide sites in the glass structure