The effect of β-cyclocitral treatment on the carotenoid content of transgenic Marsh grapefruit (Citrus paradisi Macf.) suspension-cultured cells

Zheng, Xiongjie, Zhu, Kaijie, Ye, Junli, Price, Elliott J., Deng, Xiuxin, Fraser, Paul D. (2020): The effect of β-cyclocitral treatment on the carotenoid content of transgenic Marsh grapefruit (Citrus paradisi Macf.) suspension-cultured cells. Phytochemistry (112509) 180: 1-8, DOI: 10.1016/j.phytochem.2020.112509, URL: http://dx.doi.org/10.1016/j.phytochem.2020.11250

High-resolution interrogation of functional elements in the noncoding genome

The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ∼18,000 single guide RNAs targeting > 700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.National Institutes of Health (U.S.) (Award F32-DK096822)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

The proteome of Nicotiana benthamiana is shaped by extensive protein processing

Summary: Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in‐gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor‐like kinases. Transient expression of double‐tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana

Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant U54 CA151884)National Cancer Institute (U.S.) (Grant P30-CA14051)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01-DK097768

Using Transcriptome Analysis to Identify Genes Involved in Switchgrass Flower Reversion

Floral reversion is a process in which differentiated floral organs revert back to vegetative organs. Although this phenomenon has been described for decades, the underlying molecular mechanisms remain unclear. In this study, we found that immature switchgrass (Panicum virgatum) inflorescences can revert to neonatal shoots when incubated on a basal medium with benzylaminopurine. We used anatomical and histological methods to verify that these shoots were formed from floret primordia through flower reversion. To further explore the gene regulation of floral reversion in switchgrass, the transcriptome of reversed, unreversed, and uncultured immature inflorescences were analyzed and 517 genes were identified as participating in flower reversion. Annotation using non-redundant databases revealed that these genes are involved in plant hormone biosynthesis and signal transduction, starch and sucrose metabolism, DNA replication and modification, and other processes crucial for switchgrass flower reversion. When four of the genes were overexpressed in Arabidopsis thaliana, vegetative growth was facilitated and reproductive growth was inhibited in transgenic plants. This study provides a basic understanding of genes regulating the floral transition in switchgrass and will promote the research of floral reversion and flower maintenance

Emergent Spin Phenomena in Air-Stable, Atomically Thin Lead

A stable platform to synthesize ultrathin heavy metals, with a strong interfacial Rashba effect, could lead to high efficiency charge-to-spin conversion for next-generation spintronics. Here we report wafer-scale synthesis of air-stable, epitaxially registered monolayer Pb on SiC (0001) via confinement heteroepitaxy (CHet). The highly asymmetric interfacial bonding in this heavy metal system lends to strong Rashba spin-orbit coupling near the Fermi level. Additionally, the system's air stability enables ex-situ spin torque ferromagnetic resonance (ST-FMR) measurements that demonstrate charge-to-spin conversion in CHet-based 2D-Pb/ferromagnet heterostructures and a 1.5x increase in the effective field ratio compared to control samples.Comment: 17 pages, 4 figures. Supporting Information included (20 pages, 9 figures, 1 table

Genome-wide association studies and expression-based quantitative trait loci analyses reveal roles of HCT2 in caffeoylquinic acid biosynthesis and its regulation by defense-responsive transcription factors in Populus.

3-O-caffeoylquinic acid, also known as chlorogenic acid (CGA), functions as an intermediate in lignin biosynthesis in the phenylpropanoid pathway. It is widely distributed among numerous plant species and acts as an antioxidant in both plants and animals. Using GC-MS, we discovered consistent and extreme variation in CGA content across a population of 739 4-yr-old Populus trichocarpa accessions. We performed genome-wide association studies (GWAS) from 917 P. trichocarpa accessions and expression-based quantitative trait loci (eQTL) analyses to identify key regulators. The GWAS and eQTL analyses resolved an overlapped interval encompassing a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase 2 (PtHCT2) that was significantly associated with CGA and partially characterized metabolite abundances. PtHCT2 leaf expression was significantly correlated with CGA abundance and it was regulated by cis-eQTLs containing W-box for WRKY binding. Among all nine PtHCT homologs, PtHCT2 is the only one that responds to infection by the fungal pathogen Sphaerulina musiva (a Populus pathogen). Validation using protoplast-based transient expression system suggests that PtHCT2 is regulated by the defense-responsive WRKY. These results are consistent with reports of CGA functioning as an antioxidant in response to biotic stress. This study provides insights into data-driven and omics-based inference of gene function in woody species

Efficacy of cement stabilisation for ultra-soft clay

As the need for land in Singapore rises, new land reclamation projects are moving closer to the outer areas of Singapore. Offshore land reclamation is very challenging when there is lack of granular fill and the ultra-soft soil such as dredged clay slurry has to be used. The dredged clay slurry usually has a very high water content, e.g. can be higher than twice of its liquid limit. Due to the extremely high water content, some traditional ground improvement methods, such as chemical stabilization using ordinary Portland cement (PC), may be not very cost effective for the dredged clay slurry.Ground granulated blastfurnace slag (GGBS) is a material that has come into the spotlight recently as an alternative to PC in terms of soil improvement. In order to strengthen and reach its full activation, an activator known as carbide slag (CS) is added together with GGBS. In this report the use of GGBS and CS is investigated and its effect on soil with very high water content is studied at different curing ages and binder ratios. This report also seeks the optimum ratio of CS/GGBS to benefit the cost factor for use in actual sites.Bachelor of Engineering (Civil

Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis

Auxin regulates nearly all aspects of plant growth and development including cell division, cell elongation and cell differentiation, which are achieved largely by rapid regulation of auxin response genes. However, the functions of a large number of auxin response genes remain uncharacterized. Paclobutrazol Resistance (PRE) proteins are non-DNA binding basic helix-loop-helix transcription factors that have been shown to be involved in gibberellin and brassinosteroid signaling, and light responses in Arabidopsis. Here, we provide molecular and genetic evidence that PRE6, one of the six PRE genes in Arabidopsis, is an auxin response gene, and that PRE6 is involved in the regulation of auxin signaling. By using quantitative RT-PCR, we showed that the expression level of PRE6 was increased in response to exogenously applied IAA. GUS staining results also showed that the expression of GUS reporter gene in the PRE6p:GUS transgenic seedlings was elevated in response to auxin. Phenotypic analysis showed that overexpression of PRE6 in Arabidopsis resulted in auxin-related phenotypes including elongated hypocotyl and primary roots, and reduced number of lateral roots when compared with the Col wild type seedlings, whereas opposite phenotypes were observed in the pre6 mutants. Further analysis showed that PRE6 overexpression plants were hyposensitive, whereas pre6 mutants were hypersensitive to auxin in root and hypocotyl elongation and lateral root formation assays. By using protoplasts transfection, we showed that PRE6 functions as a transcriptional repressor. Consistent with this, the expression of the auxin response reporter DR5:GUS was decreased in PRE6 overexpression lines, but increased in pre6 mutants. When co-transfected into protoplasts, ARF5 and ARF8 activated the expression of the PRE6p:GUS reporter. Chromatin immunoprecipitation assays showed that ARF5 and ARF8 can be recruited to the promoter regions of PRE6. Taken together, these results suggest that PRE6 is an auxin response gene whose expression is directly regulated by ARF5 and ARF8, and that PRE6 is a transcriptional repressor that negatively regulates auxin responses in Arabidopsis