@article{gentzel_park_bellizzi_xiao_gadhave_murphree_yang_lamantia_redinbaugh_balint-kurti_et al._2020, title={A CRISPR/dCas9 toolkit for functional analysis of maize genes}, volume={16}, ISSN={1746-4811}, url={http://dx.doi.org/10.1186/s13007-020-00675-5}, DOI={10.1186/s13007-020-00675-5}, abstractNote={The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9).In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression.We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.}, number={1}, journal={Plant Methods}, publisher={Springer Science and Business Media LLC}, author={Gentzel, Irene N. and Park, Chan Ho and Bellizzi, Maria and Xiao, Guiqing and Gadhave, Kiran R. and Murphree, Colin and Yang, Qin and LaMantia, Jonathan and Redinbaugh, Margaret G. and Balint-Kurti, Peter and et al.}, year={2020}, month={Oct} }
 @article{dums_murphree_vasani_young_sederoff_2018, title={Metabolic and Transcriptional Profiles of Dunaliella viridis Supplemented With Ammonium Derived From Glutamine}, volume={5}, ISSN={["2296-7745"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85052906510&partnerID=MN8TOARS}, DOI={10.3389/fmars.2018.00311}, abstractNote={Algal biofuel production requires an input of synthetic nitrogen fertilizer. Fertilizer synthesized via the Haber-Bosch process produces CO2 as a waste byproduct and represents a substantial financial and energy investment. Reliance on synthetic fertilizer attenuates the environmental significance and economic viability of algae production systems. To lower fertilizer input, the waste streams of algal production systems can be recycled to provide alternative sources of nitrogen such as amino acids to the algae. The halophytic green alga Dunaliella viridis can use ammonium (NH4+) derived from the abiotic degradation of amino acids, and previously, supplementation of NH4+ from glutamine degradation was shown to support acceptable levels of growth and increased neutral lipid production compared to nitrate. To understand the effect of glutamine-released NH4+ on algae growth and physiology, metabolite levels, growth parameters, and transcript profiles of D. viridis cultures were observed in a time course after transition from media containing nitrate as a sole N source to medium containing glutamine, glutamate, or a N-depleted medium. Growth parameters were similar between glutamine (NH4+) and nitrate supplemented cultures, however, metabolite data showed that the glutamine supplemented cultures (NH4+) more closely resembled cultures under nitrogen starvation (N-depleted and glutamate supplementation). Neutral lipid accumulation was the same in nitrate and glutamine-derived NH4+ cultures. However, glutamine-derived NH4+ caused a transcriptional response in the immediate hours after inoculation of the culture. The strong initial response of cultures to NH4+ changed over the course of days to closely resemble that of nitrogen starvation. These observations suggest that release of NH4+ from glutamine was sufficient to maintain growth, but not high enough to trigger a cell transition to a nitrogen replete state. Comparative transcript profiling of the nitrogen-starved and nitrate-supplied cultures show an overall downregulation of fatty acid synthesis and a shift to starch synthesis and accumulation. The results indicate that a continuous, amino acid derived slow release of NH4+ to algae cultures could reduce the amount of synthetic nitrogen needed for growth, but optimization is needed to balance nitrogen starvation and cell division.}, number={AUG}, journal={FRONTIERS IN MARINE SCIENCE}, author={Dums, Jacob and Murphree, Colin and Vasani, Naresh and Young, Danielle and Sederoff, Heike}, year={2018}, month={Aug} }
 @article{murphree_dums_jain_zhao_young_khoshnoodi_tikunov_macdonald_pilot_sederoff_et al._2017, title={Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth}, volume={8}, ISSN={["1664-462X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85021390666&partnerID=MN8TOARS}, DOI={10.3389/fpls.2017.00847}, abstractNote={Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Murphree, C. A. and Dums, J. T. and Jain, S. K. and Zhao, C. S. and Young, D. Y. and Khoshnoodi, N. and Tikunov, A. and Macdonald, J. and Pilot, G. and Sederoff, Heike and et al.}, year={2017}, month={May} }