Publications

2015
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species.. PloS one. 10(10):e0140285. Abstract
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
Chakraborty, A, Mazumder A, Lin M, Hasemeyer A, Xu Q, Wang D, Ebright YW, Ebright RH.  2015.  Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.. Methods in molecular biology (Clifton, N.J.). 1276:101-31. Abstract
A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.
Galli, M, Liu Q, Moss BL, Malcomber S, Li W, Gaines C, Federici S, Roshkovan J, Meeley R, Nemhauser J et al..  2015.  Auxin signaling modules regulate maize inflorescence architecture. Proc Natl Acad Sci USA. 112:13372-13377. AbstractWebsite
In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.
Zhang, Y, Wang P, Shao W, Zhu J-K, Dong J.  2015.  The BASL Polarity Protein Controls a MAPK Signaling Feedback Loop in Asymmetric Cell Division.. Dev Cell. doi: 10.1016/j.devcel.2015.02.022.
Druzhinin, SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE.  2015.  A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq. PLoS Genet. 11(7):e1005348.
Ambegaonkar, AA, Irvine KD.  2015.  Coordination of planar cell polarity pathways through Spiny legs. eLife. 4:pii:e09946.
Singaravelu, G, Rahimi S, Krauchunas A, Rizvi A, Dharia S, Shakes D, Smith H, Golden A, Singson A.  2015.  Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization.. Current Biology. 25:3220-3224.
Cruz, JW, Sharp JD, Hoffer ED, Maehigashi T, Vvedenskaya IO, Konkimalla A, Husson RN, Nickels BE, Dunham C, Woychik NA.  2015.  Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun. 6:7480.
Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
Radford, SJ, Hoang TL, Gluszek AA, Ohkura H, McKim KS.  2015.  Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS Genetics. 11(10):e1005605.Website
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively systematic transcript end readout, “MASTER”: transcription start site selection, transcriptional slippage, and transcript yields. Molecular Cell. 60:953-965.
Akhurst, RJ, Padgett RW.  2015.  Matters of context guide future research in TGFβ superfamily signaling. Science Signaling. 8(399):DOI:10.1126/scisignal.aad0416.
Krishnan, A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev GM, Posewitz MZ, Dismukes GC.  2015.  Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.. Plant J. 81(6):947-960. Abstract
Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.
Gallavotti, A. and Whipple, CJ.  2015.  Positional cloning in maize (Zea mays subsp. mays, Poaceae). Applications in Plant Sciences. 3:1400092.Website
Vvedenskaya, IO, Goldman SR, Nickels BE.  2015.  Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5' ends. Methods Mol Biol. 1276:211-228.
Goldman, SR, Nair N, Wells C, Nickels BE, Hochschild A.  2015.  The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo. eLife. 4:e10514.
Gardner, G, Al-Sharab J, Danilovic N, Go Y B, Ayers KE, Greenblatt M, Dismukes G C.  2015.  Structural Basis for Differing Electrocatalytic Water Oxidation by the Cubic, Layered and Spinel Forms of Lithium Cobalt Oxides. Energy Environ. Sci.. :-. AbstractWebsite
The two polymorphs of lithium cobalt oxide, LiCoO2, present an opportunity to contrast the structural requirements for reversible charge storage (battery function) vs catalysis of water oxidation/oxygen evolution (OER; 2H2O[rightward arrow]O2 + 4H+ + 4e- ). Previously, we reported high OER electrocatalytic activity from nanocrystals of the cubic phase vs. poor activity from the layered phase - the archetypal lithium-ion battery cathode. Here we apply transmission electron microscopy, electron diffraction, voltammetry and elemental analysis under OER electrolysis condition to show that labile Li+ ions (de)intercalate from layered LiCoO2, initiating structural reorganization to the cubic spinel LiCo2O4, in parallel with formation of an active catalytic phase. Comparison of cubic LiCoO2 (50nm) to iridium (5 nm) nanoparticles for OER catalysis (commercial benchmark) in basic and neutral electrolyte reveals excellent performance in terms of Tafel slope (48 mV dec-1), overpotential ([small eta] =  420 mV @ 10 mA cm-2 at pH = 14), Faradic yield (100%) and OER stability (no loss in 14 hours). The inherent OER activity of cubic LiCoO2 and spinel LiCo2O4 is attributable to their [Co4O4]n+ cubane structural units, which provides lower oxidation potential to Co4+ and lower inter-cubane hole mobility. By contrast, the layered phase which lacks cubanes exhibits extensive intra-planar hole delocalization which entropically disfavors the four electron/hole concerted OER reaction.
Deibert, BJ, Zhang J, Smith PF, Chapman KW, Rangan S, Banerjee D, Tan K, Wang H, Pasguale N, Chen F et al..  2015.  Surface and Structural Investigation of a MnOx Birnessite-Type Water Oxidation Catalyst Formed under Photocatalytic Conditions. Chemistry. 21(40):14218-14228. Abstract
Catalytically active MnOx species have been reported to form in situ from various Mn-complexes during electrocatalytic and solution-based water oxidation when employing cerium(IV) ammonium ammonium nitrate (CAN) oxidant as a sacrificial reagent. The full structural characterization of these oxides may be complicated by the presence of support material and lack of a pure bulk phase. For the first time, we show that highly active MnOx catalysts form without supports in situ under photocatalytic conditions. Our most active (4)MnOx catalyst (∼0.84 mmol O2  mol Mn(-1) s(-1)) forms from a Mn4O4 bearing a metal-organic framework. (4)MnOx is characterized by pair distribution function analysis (PDF), Raman spectroscopy, and HR-TEM as a disordered, layered Mn-oxide with high surface area (216 m(2) g(-1)) and small regions of crystallinity and layer flexibility. In contrast, the (S)MnOx formed from Mn(2+) salt gives an amorphous species of lower surface area (80 m(2) g(-1)) and lower activity (∼0.15 mmol O2  mol Mn(-1) s(-1)). We compare these catalysts to crystalline hexagonal birnessite, which activates under the same conditions. Full deconvolution of the XPS Mn2p3/2 core levels detects enriched Mn(3+) and Mn(2+) content on the surfaces, which indicates possible disproportionation/comproportionation surface equilibria.
Ramsey, KM, Osborne ML, Vvedenskaya IO, Su C, Nickels BE, Dove SL.  2015.  Ubiquitous promoter-localization of essential virulence regulators in Francisella tularensis.. PLoS Pathog. 11(4):e1004793.
Smith, PF, Hunt L, Laursen AB, Sagar V, Kaushik S, Calvinho KU, Marotta G, Mosconi E, De Angelis F, Dismukes GC.  2015.  Water Oxidation by the [Co4O4(OAc)4(py)4](+) Cubium is Initiated by OH(-) Addition.. J Am Chem Soc. 137(49):15460-15468. Abstract
The cobalt cubium Co4O4(OAc)4(py)4(ClO4) (1A(+)) containing the mixed valence [Co4O4](5+) core is shown by multiple spectroscopic methods to react with hydroxide (OH(-)) but not with water molecules to produce O2. The yield of reaction products is stoichiometric (>99.5%): 41A(+) + 4OH(-) → O2 + 2H2O + 41A. By contrast, the structurally homologous cubium Co4O4(trans-OAc)2(bpy)4(ClO4)3, 1B(ClO4)3, produces no O2. EPR/NMR spectroscopies show clean conversion to cubane 1A during O2 evolution with no Co(2+) or Co3O4 side products. Mass spectrometry of the reaction between isotopically labeled μ-(16)O(bridging-oxo) 1A(+) and (18)O-bicarbonate/water shows (1) no exchange of (18)O into the bridging oxos of 1A(+), and (2) (36)O2 is the major product, thus requiring two OH(-) in the reactive intermediate. DFT calculations of solvated intermediates suggest that addition of two OH(-) to 1A(+) via OH(-) insertion into Co-OAc bonds is energetically favored, followed by outer-sphere oxidation to intermediate [1A(OH)2](0). The absence of O2 production by cubium 1B(3+) indicates the reactive intermediate derived from 1A(+) requires gem-1,1-dihydoxo stereochemistry to perform O-O bond formation. Outer-sphere oxidation of this intermediate by 2 equiv of 1A(+) accounts for the final stoichiometry. Collectively, these results and recent literature (Faraday Discuss., doi:10.1039/C5FD00076A and J. Am. Chem. Soc. 2015, 137, 12865-12872) validate the [Co4O4](4+/5+) cubane core as an intrinsic catalyst for oxidation of hydroxide by an inner-sphere mechanism.
Sun, S, Reddy BVVG, Irvine KD.  2015.  Localization of Hippo Signaling complexes and Warts activation in vivo. Nature Communications. 6:8402.
Durst, R, Peal DS, deVlaming A, Leyne M, Talkowski M, Perrocheau M, Simpson C, Jett C, Stone MR, Charles F et al..  2015.  Mutations in DCHS1 Cause Mitral Valve Prolapse. Nature. :inpress.
Wang, W, Zhang W, Wu Y, Maliga P, Messing J.  2015.  RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species. PLoS One. 10:e0140285. AbstractWebsite
RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.
2014
Lang, Z, Wills DM, Lemmon ZH, Shannon LM, Bukowski R, Wu Y, Messing J, Doebley JF.  2014.  Defining the Role of prolamin-box binding factor1 Gene During Maize Domestication. J Hered. AbstractWebsite
The prolamin-box binding factor1 (pbf1) gene encodes a transcription factor that controls the expression of seed storage protein (zein) genes in maize. Prior studies show that pbf1 underwent selection during maize domestication although how it affected trait change during domestication is unknown. To assay how pbf1 affects phenotypic differences between maize and teosinte, we compared nearly isogenic lines (NILs) that differ for a maize versus teosinte allele of pbf1. Kernel weight for the teosinte NIL (162mg) is slightly but significantly greater than that for the maize NIL (156mg). RNAseq data for developing kernels show that the teosinte allele of pbf1 is expressed at about twice the level of the maize allele. However, RNA and protein assays showed no difference in zein profile between the two NILs. The lower expression for the maize pbf1 allele suggests that selection may have favored this change; however, how reduced pbf1 expression alters phenotype remains unknown. One possibility is that pbf1 regulates genes other than zeins and thereby is a domestication trait. The observed drop in seed weight associated with the maize allele of pbf1 is counterintuitive but could represent a negative pleiotropic effect of selection on some other aspect of kernel composition.