Krishnan, A, Zhang S, Liu Y, Tadmori KA, Bryant DA, Dismukes GC.  2016.  Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. Biotechnol Bioeng. Abstract
CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells-1 h-1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.
Kane, NS, Vora M, Varre KJ, Padgett RW.  2016.  Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila using a co-CRISPR Strategy. G3. 7(1):87-93.
Galli, M and Gallavotti, A.  2016.  Expanding the regulatory network for meristem size in plants. Trends in Genetics. 32(6):372-383. AbstractWebsite
The remarkable plasticity of post-embryonic plant development is due to groups of stem-cell-containing structures called meristems. In the shoot, meristems continuously produce organs such as leaves, flowers, and stems. Nearly two decades ago the WUSCHEL/CLAVATA (WUS/CLV) negative feedback loop was established as being essential for regulating the size of shoot meristems by maintaining a delicate balance between stem cell proliferation and cell recruitment for the differentiation of lateral primordia. Recent research in various model species (Arabidopsis, tomato, maize, and rice) has led to discoveries of additional components that further refine and improve the current model of meristem regu- lation, adding new complexity to a vital network for plant growth and productivity.
Zhang, Y, Bergmann DC, Dong J.  2016.  Fine-scale dissection of the subdomains of polarity protein BASL in stomatal asymmetric cell division.. J. Exp. Bot.. doi:10.1093/jxb/erw274
Qian, X, Kim M K, Kumaraswamy KG, Agarwal A, Lun DS, Dismukes CG.  2016.  Flux balance analysis of photoautotrophic metabolism: Uncovering new biological details of subsystems involved in cyanobacterial photosynthesis. Biochimica et Biophysica Acta (BBA) - Bioenergetics. :-. AbstractWebsite
We have constructed and experimentally tested a comprehensive genome-scale model of photoautotrophic growth, denoted iSyp821, for the cyanobacterium Synechococcus sp. PCC 7002. iSyp821 incorporates a variable biomass objective function (vBOF), in which stoichiometries of the major biomass components vary according to light intensity. The vBOF was constrained to fit the measured cellular carbohydrate/protein content under different light intensities. iSyp821 provides rigorous agreement with experimentally measured cell growth rates and inorganic carbon uptake rates as a function of light intensity. iSyp821 predicts two observed metabolic transitions that occur as light intensity increases: 1) from PSI-cyclic to linear electron flow (greater redox energy), and 2) from carbon allocation as proteins (growth) to carbohydrates (energy storage) mode. iSyp821 predicts photoautotrophic carbon flux into 1) a hybrid gluconeogenesis-pentose phosphate (PP) pathway that produces glycogen by an alternative pathway than conventional gluconeogenesis, and 2) the photorespiration pathway to synthesize the essential amino acid, glycine. Quantitative fluxes through both pathways were verified experimentally by following the kinetics of formation of 13C metabolites from 13CO2 fixation. iSyp821 was modified to include changes in gene products (enzymes) from experimentally measured transcriptomic data and applied to estimate changes in concentrations of metabolites arising from nutrient stress. Using this strategy, we found that iSyp821 correctly predicts the observed redistribution pattern of carbon products under nitrogen depletion, including decreased rates of CO2 uptake, amino acid synthesis, and increased rates of glycogen and lipid synthesis.
Vvedenskaya, IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE.  2016.  Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.. Proc. Natl. Acad. Sci. U.S.A. 113(21):E2899-2905.
Xue, X, Dong J.  2016.  The MAPK substrate MASS2 regulates stomatal patterning in Arabidopsis.. Mol. Reprod. Dev. . 83(2):93.
Hou, Y, Guo X, Cyprys P, Zhang Y, Bleckmann A, Cai L, Huang Q, Luo Y, Gu H, Dresselhaus T et al..  2016.  Maternal ENODLs Are Required for Pollen Tube Reception in Arabidopsis.. Curr. Biol.. doi:10.1016/j.cub.2016.06.053
Bird, JG, Zhang Y, Tian Y, Panova N, Barvík I, Greene L, Liu M, Buckley B, Krásný L, Lee JK et al..  2016.  The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA. Nature. 535(7612):444-447.
Krauchunas, AR, Marcello MR, Singson "A.  2016.  The molecular complexity of fertilization: Introducing the concept of a fertilization synapse. Molecular Reproduction and Development.
Winkelman, JT, Vvedenskaya IO, Zhang Y, Zhang Y, Bird JG, Taylor DM, Gourse RL, Ebright RH, Nickels BE.  2016.  Multiplexed protein-DNA crosslinking: scrunching in transcription start site selection. Science. 351(6277):1090-1093.
Vinyard, DJ, Sun JS, Gimpel J, Ananyev GM, Mayfield SP, Dismukes GC.  2016.  Natural isoforms of the Photosystem II D1 subunit differ in photoassembly efficiency of the water-oxidizing complex.. Photosynth Res.. Abstract
Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn2+, Ca2+, water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn3+ intermediate, IM1*, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680 + Q A - ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680 + Q A - ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity.
Ananyev, GM, Gates C, Dismukes GC.  2016.  The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.. Biochim Biophys Acta.. 1857(9):1380-1391. Abstract
We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB- and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB- acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.
Zhang, W, Messing J.  2016.  PacBio for Haplotyping in Gene Families. Haplotyping. Methods in Molecular Biology 1551. :61-71.
Shao, W, Dong J.  2016.  Polarity in plant asymmetric cell division: Division orientation and cell fate differentiation.. Dev. Biol.. doi:10.1016/j.ydbio.2016.07.020
Gates, C, Ananyev GM, Dismukes C.  2016.  The strontium inorganic mutant of the water oxidizing center (CaMn4O5) of PSII improves WOC efficiency but slows electron flux through the terminal acceptors.. Biochim Biophys Acta.. 1857(9):1550-1560. Abstract
Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5–8 × slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45 °C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S = 5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S = 1/2 form.
Delatte, B, Wang F, Ngoc L V, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S et al..  2016.  Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science. 351(6270):282-285. AbstractWebsite
Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.
Schifano, JM, Cruz JW, Edifor R, Vvedenskaya IO, Ouyang M, Husson RN, Nickels BE, Woychik NA.  2016.  tRNA is a new target for cleavage by a MazF toxin. Nucleic Acids Research. 44(3):1256-1270.
Lutz, KA, Martin C, Khairzada S, Maliga P.  2015.  Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture. Plant Cell Rep. 34:1849-56. AbstractWebsite
KEY MESSAGE: We describe a steroid-inducible BABY BOOM system that improves plant regeneration in Arabidopsis leaf cultures and yields fertile plants. Regeneration of Arabidopsis thaliana plants for extended periods of time in tissue culture may result in sterile plants. We report here a novel approach for A. thaliana regeneration using a regulated system to induce embryogenic cultures from leaf tissue. The system is based on BABY BOOM (BBM), a transcription factor that turns on genes involved in embryogenesis. We transformed the nucleus of A. thaliana plants with BBM:GR, a gene in which the BBM coding region is fused with the glucocorticoid receptor (GR) steroid-binding domain. In the absence of the synthetic steroid dexamethasone (DEX), the BBM:GR fusion protein is localized in the cytoplasm. Only when DEX is included in the culture medium does the BBM transcription factor enter the nucleus and turn on genes involved in embryogenesis. BBM:GR plant lines show prolific shoot regeneration from leaf pieces on media containing DEX. Removal of DEX from the culture media allowed for flowering and seed formation. Therefore, use of BBM:GR leaf tissue for regeneration of plants for extended periods of time in tissue culture will facilitate the recovery of fertile plants.
Mao, Y, Francis-West P, Irvine KD.  2015.  A Fat4-Dchs1 signal between stromal and cap mesenchyme cells influences nephrogenesis and ureteric bud branching.. Development (Cambridge, England). AbstractWebsite
Formation of the kidney requires reciprocal signaling among the ureteric tubules, cap mesenchyme and surrounding stromal mesenchyme to orchestrate complex morphogenetic events. The protocadherin Fat4 influences signaling from stromal to cap mesenchyme cells to influence their differentiation into nephrons. Here we characterize the role of a putative binding partner of Fat4, the protocadherin Dchs1. Mutation of Dchs1 leads to increased numbers of cap mesenchyme cells, which are abnormally arranged around the ureteric bud tips, and impairs nephron morphogenesis. Mutation of Dchs1 also reduces branching of the ureteric bud and impairs differentiation of ureteric bud tip cells into trunk cells. Genetically, Dchs1 is required specifically within cap mesenschyme cells. The similarity of Dchs1 phenotypes to stromal-less kidneys and to Fat4 mutants implicate Dchs1 in Fat4-dependent stroma-to-cap mesenchyme signaling. Antibody staining of genetic mosaics reveals that Dchs1 protein localization is polarized within cap mesenchyme cells, where it accumulates at the interface with stromal cells, implying that it interacts directly with a stromal protein. Our observations identify a role for Fat4-Dchs1 in signaling between cell layers, implicate Dchs1 as a Fat4 receptor for stromal signaling that is essential for kidney development, and establish that vertebrate Dchs1 can be molecularly polarized in vivo.
Irvine, KD, Harvey KF.  2015.  Control of organ growth by patterning and hippo signaling in Drosophila.. Cold Spring Harbor perspectives in biology. 7 AbstractWebsite
Control of organ size is of fundamental importance and is controlled by genetic, environmental, and mechanical factors. Studies in many species have pointed to the existence of both organ-extrinsic and -intrinsic size-control mechanisms, which ultimately must coordinate to regulate organ size. Here, we discuss organ size control by organ patterning and the Hippo pathway, which both act in an organ-intrinsic fashion. The influence of morphogens and other patterning molecules couples growth and patterning, whereas emerging evidence suggests that the Hippo pathway controls growth in response to mechanical stimuli and signals emanating from cell-cell interactions. Several points of cross talk have been reported between signaling pathways that control organ patterning and the Hippo pathway, both at the level of membrane receptors and transcriptional regulators. However, despite substantial progress in the past decade, key questions in the growth-control field remain, including precisely how and when organ patterning and the Hippo pathway communicate to control size, and whether these communication mechanisms are organ specific or general. In addition, elucidating mechanisms by which organ-intrinsic cues, such as patterning factors and the Hippo pathway, interface with extrinsic cues, such as hormones to control organ size, remain unresolved.
Cao, HX, Vu GT, Wang W, Messing J, Schubert I.  2015.  Chromatin organisation in duckweed interphase nuclei in relation to the nuclear DNA content. Plant Biol (Stuttg). 17 Suppl 1:120-4. AbstractWebsite
The accessibility of DNA during fundamental processes, such as transcription, replication and DNA repair, is tightly modulated through a dynamic chromatin structure. Differences in large-scale chromatin structure at the microscopic level can be observed as euchromatic and heterochromatic domains in interphase nuclei. Here, key epigenetic marks, including histone H3 methylation and 5-methylcytosine (5-mC) as a DNA modification, were studied cytologically to describe the chromatin organisation of representative species of the five duckweed genera in the context of their nuclear DNA content, which ranged from 158 to 1881 Mbp. All studied duckweeds, including Spirodela polyrhiza with a genome size and repeat proportion similar to that of Arabidopsis thaliana, showed dispersed distribution of heterochromatin signatures (5mC, H3K9me2 and H3K27me1). This immunolabelling pattern resembles that of early developmental stages of Arabidopsis nuclei, with less pronounced heterochromatin chromocenters and heterochromatic marks weakly dispersed throughout the nucleus.
Wang, W, Messing J.  2015.  Status of duckweed genomics and transcriptomics. Plant Biol (Stuttg). 17 Suppl 1:10-5. AbstractWebsite
Duckweeds belong to the smallest flowering plants that undergo fast vegetative growth in an aquatic environment. They are commonly used in wastewater treatment and animal feed. Whereas duckweeds have been studied at the biochemical level, their reduced morphology and wide environmental adaption had not been subjected to molecular analysis until recently. Here, we review the progress that has been made in using a DNA barcode system and the sequences of chloroplast and mitochondrial genomes to identify duckweed species at the species or population level. We also review analysis of the nuclear genome sequence of Spirodela that provides new insights into fundamental biological questions. Indeed, reduced gene families and missing genes are consistent with its compact morphogenesis, aquatic floating and suppression of juvenile-to-adult transition. Furthermore, deep RNA sequencing of Spirodela at the onset of dormancy and Landoltia in exposure of nutrient deficiency illustrate the molecular network for environmental adaption and stress response, constituting major progress towards a post-genome sequencing phase, where further functional genomic details can be explored. Rapid advances in sequencing technologies could continue to promote a proliferation of genome sequences for additional ecotypes as well as for other duckweed species.