Hawkins, JS, Delgado V, Feng L, Carlise M, Dooner HK, Bennetzen JL.  2014.  Variation in allelic expression associated with a recombination hotspot in Zea mays.. The Plant Journal, DOI: 10.1111/tpj.12537. Abstract
Gene expression is a complex process, requiring precise spatial and temporal regulation of transcription factor activity; however, modifications of individual cis- and trans-acting modules can be molded by natural selection to create a sizeable number of novel phenotypes. Results from decades of research indicate that developmental and phenotypic divergence among eukaryotic organisms is driven primarily by variation in levels of gene expression that are dictated by mutations either in structural or regulatory regions of genes. The relative contributions and interplay of cis- and trans-acting regulatory factors to this evolutionary process, however, remain poorly understood. Analysis of 8 genes in the Bz1-Sh1 interval of maize indicates significant allele-specific expression biases in at least one tissue for all genes, ranging from 1.3-fold to 36-fold. All detected effects were cis-regulatory in nature, although genetic background may also influence the level of expression bias and tissue specificity for some allelic combinations. Most allelic pairs exhibited the same direction and approximate intensity of bias across all four tissues; however, a subset of allelic pairs show alternating dominance across different tissue types or variation in the degree of bias in different tissues. In addition, the genes showing the most striking levels of allelic bias co-localize with a previously described recombination hotspot in this region, suggesting a naturally occurring genetic mechanism for creating regulatory variability for a subset of plant genes that may ultimately lead to evolutionary diversification.
Zhang, Y, Degen D, Ho MX, Sineva E, Ebright KY, Ebright YW, Mekler V, Vahedian-Movahed H, Feng Y, Yin R et al..  2014.  GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.. eLife. 3:e02450. Abstract
Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI:
Gleason, RJ, Akintobi AM, Grant BD, Padgett RW.  2014.  BMP signaling requires retromer-dependent recycling of the type I receptor. Proc. Natl. Acad. Sci., USA . 10.1073/pnas.1319947111
Chatterjee, M, Tabi Z, Galli M, Malcomber S, Buck A, Muszynski M, Gallavotti A.  2014.  The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility. Plant Cell. (26):2962-2977. AbstractWebsite
Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development.We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.
Maliga, P.  2014.  Chloroplast Biotechnology: Methods and Protocols. Methods in Molecular Biology. 1132Website
Xiong, W, He L, Lai J, Dooner HK, Du C.  2014.  HelitronScanner uncovers a large overlooked cache of Helitron transposons in many genomes.. Proc. Natl. Acad. Sci. USA. DOI 10.1073/pnas.1410068111 AbstractWebsite
Transposons make up the bulk of eukaryotic genomes, but are difficult to annotate because they evolve rapidly. Most of the unannotated portion of sequenced genomes is probably made up of various divergent transposons that have yet to be categorized. Helitrons are unusual rolling circle eukaryotic transposons that often capture gene sequences, making them of considerable evolutionary importance. Unlike other DNA transposons, Helitrons do not end in inverted repeats or create target site duplications, so they are particularly challenging to identify. Here we present HelitronScanner, a two-layered local combinational variable (LCV) tool for generalized Helitron identification that represents a major improvement over previous identification programs based on DNA sequence or structure. HelitronScanner identified 64,654 Helitrons from a wide range of plant genomes in a highly automated way. We tested HelitronScanner’s predictive ability in maize, a species with highly heterogeneous Helitron elements. LCV scores for the 5’ and 3’ termini of the predicted Helitrons provide a primary confidence level and element copy number provides a secondary one. Newly identified Helitrons were validated by polymerase chain reaction (PCR) assays or by in-silico comparative analysis of insertion site polymorphism among multiple accessions. Many new Helitrons were identified in model species, such as maize, rice, and Arabidopsis, and in a variety of organisms where Helitrons had not been reported previously, leading to a major upward reassessment of their abundance in plant genomes. HelitronScanner promises to be a valuable tool in future comparative and evolutionary studies of this major transposon superfamily.
Nasr, I, Messing J, Ciclitira PJ.  2014.  Novel and Experimental Therapies on the Horizon. Celiac Disease, Clinical Gastroenterology. :193-208.
Yamamuro, C, Miki D, Zheng Z, Wang J, Dong J, Zhu JK.  2014.  Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.. Nature Commun.. 5(5):4062.
Tungsuchat-Huang, T, Maliga P.  2014.  Plastid marker gene excision in greenhouse-grown tobacco by Agrobacterium-delivered Cre recombinase. Chloroplast Biotechnology. 1132:205-220. Abstract
Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar au gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar au marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein.
Maliga, P, Tungsuchat-Huang T.  2014.  Plastid transformation in Nicotiana tabacum and Nicotiana sylvestris by biolistic DNA delivery to leaves. Chloroplast Biotechnology: Methods and Protocols. 1132:147-163. Abstract
The protocol we report here is based on biolistic delivery of the transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. Also, the tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar, allowing plastid engineering protocols developed for N. tabacum to be directly applied to N. sylvestris. However, the tissue culture protocol is applicable only in a subset of N. tabacum cultivars. Here we highlight differences between the protocols for the two species. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome as well as systems for marker excision. The simpler genetics of the diploid N. sylvestris, as opposed to the allotetraploid N. tabacum, make it an attractive model for plastid transformation.
Wu, Y, Messing J.  2014.  Proteome balancing of the maize seed for higher nutritional value. Front Plant Sci. 5:240. AbstractWebsite
Most flowering plant seeds are composed of the embryo and endosperm, which are surrounded by maternal tissue, in particular the seed coat. Whereas the embryo is the dormant progeny, the endosperm is a terminal organ for storage of sugars and amino acids in proteins and carbohydrates, respectively. Produced in maternal leaves during photosynthesis, sugars, and amino acids are transported to developing seeds after flowering, and during germination they nourish early seedlings growth. Maize endosperm usually contains around 10% protein and 70% starch, and their composition ratio is rather stable, because it is strictly regulated through a pre-set genetic program that is woven by networks of many interacting or counteracting genes and pathways. Endosperm protein, however, is of low nutritional value due mainly to the high expression of the alpha-zein gene family, which encodes lysine-free proteins. Reduced levels of these proteins in the opaque 2 (o2) mutant and alpha-zein RNAi (RNA interference) transgenic seed is compensated by an increase of non-zein proteins, leading to the rebalancing of the nitrogen sink and producing more or less constant levels of total proteins in the seed. The same rebalancing of zeins and non-zeins has been observed for maize seeds bred for 30% protein. In contrast to the nitrogen sink, storage of sulfur is controlled through the accumulation of specialized sulfur-rich proteins in maize endosperm. Silencing the synthesis of alpha-zeins through RNAi fails to raise sulfur-rich proteins. Although overexpression of the methionine-rich delta-zein can increase the methionine level in seeds, it occurs at least in part at the expense of the cysteine-rich beta- and gamma-zeins, demonstrating a balance between cysteine and methionine in sulfur storage. Therefore, we propose that the throttle for the flow of sulfur is placed before the synthesis of sulfur amino acids when sulfur is taken up and reduced during photosynthesis.
Codelia, V, Sun G, Irvine KD.  2014.  Regulation of YAP by Mechanical Strain through Jnk and Hippo Signaling. Current Biology. 24:2012-2017.Website
Schifano, JM, Vvedenskaya IO, Knoblauch JG, Ouyang M, Nickels BE, Woychik NA.  2014.  An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3.. Nature Communications. 5:1–11. Abstract
Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited. We describe a rapid genome-scale approach, MORE (mapping by overexpression of an RNase in Escherichia coli) RNA-seq, for defining the cleavage specificity of endoribonucleolytic toxins. Application of MORE RNA-seq to MazF-mt3 from Mycobacterium tuberculosis reveals two critical ribosomal targets-the essential, evolutionarily conserved helix/loop 70 of 23S rRNA and the anti-Shine-Dalgarno (aSD) sequence of 16S rRNA. Our findings support an emerging model where both ribosomal and messenger RNAs are principal targets of MazF toxins and suggest that, as in E. coli, removal of the aSD sequence by a MazF toxin modifies ribosomes to selectively translate leaderless mRNAs in M. tuberculosis.
Wang, W, Wu Y, Messing J.  2014.  RNA-Seq transcriptome analysis of Spirodela dormancy without reproduction. BMC Genomics. 15(60) Abstract
Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications.
Wang, W, Wu Y, Messing J.  2014.  RNA-Seq transcriptome analysis of Spirodela dormancy without reproduction. BMC Genomics. 15:60. AbstractWebsite
BACKGROUND: Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications. RESULTS: We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass. CONCLUSIONS: The identification and functional annotation of differentially expressed genes open a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.
Wang, W, Haberer G, Gundlach H, Glasser C, Nussbaumer T, Luo MC, Lomsadze A, Borodovsky M, Kerstetter RA, Shanklin J et al..  2014.  The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat Commun. 5:3311. AbstractWebsite
The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.
Vorobiev, SM, Gensler Y, Vahedian-Movahed H, Seetharaman J, Su M, Huang JY, Xiao R, Kornhaber G, Montelione GT, Tong L et al..  2014.  Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor λQ. Structure. 22:488-495.
Durbak, AR, Phillips KA, Pike S, O'Neill MA, Mares J, Gallavotti A, Malcomber S, Gassmann W, McSteen P.  2014.  Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize. Plant Cell. (26):2978-2995. AbstractWebsite
The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.
Gurdon, C, Maliga P.  2014.  Two distinct plastid genome configurations and unprecedented intraspecies length variation in the accD coding region in Medicago truncatula. DNA Reserach. 21:inpress. AbstractWebsite
We fully sequenced four and partially sequenced six additional plastid genomes of the model legume Medicago truncatula. Three accessions, Jemalong 2HA, Borung and Paraggio, belong to ssp. truncatula, and R108 to ssp. tricycla. We report here that the R108 ptDNA has a ∼45-kb inversion compared with the ptDNA in ssp. truncatula, mediated by a short, imperfect repeat. DNA gel blot analyses of seven additional ssp. tricycla accessions detected only one of the two alternative genome arrangements, represented by three and four accessions each. Furthermore, we found a variable number of repeats in the essential accD and ycf1 coding regions. The repeats within accD are recombinationally active, yielding variable-length insertions and deletions in the central part of the coding region. The length of ACCD was distinct in each of the 10 sequenced ecotypes, ranging between 650 and 796 amino acids. The repeats in the ycf1 coding region are also recombinationally active, yielding short indels in 10 regions of the reading frames. Thus, the plastid genome variability we report here could be linked to repeat-mediated genome rearrangements. However, the rate of recombination was sufficiently low, so that no heterogeneity of ptDNA could be observed in populations maintained by single-seed descent.
Minakhina, S, Changela N, Steward R.  2014.  Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery.. Development. 141(2):259-268. AbstractWebsite
The maintenance of stem cells is central to generating diverse cell populations in many tissues throughout the life of an animal. Elucidating the mechanisms involved in how stem cells are formed and maintained is crucial to understanding both normal developmental processes and the growth of many cancers. Previously, we showed that Zfrp8/PDCD2 is essential for the maintenance of Drosophila hematopoietic stem cells. Here, we show that Zfrp8/PDCD2 is also required in both germline and follicle stem cells in the Drosophila ovary. Expression of human PDCD2 fully rescues the Zfrp8 phenotype, underlining the functional conservation of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis, and we find that nuclear localization of Zfrp8 in germline stem cells and their offspring is regulated by some piRNA pathway genes. We also show that Zfrp8 forms a complex with the piRNA pathway protein Maelstrom and controls the accumulation of Maelstrom in the nuage. Furthermore, Zfrp8 regulates the activity of specific transposable elements also controlled by Maelstrom and Piwi. Our results suggest that Zfrp8/PDCD2 is not an integral member of the piRNA pathway, but has an overlapping function, possibly competing with Maelstrom and Piwi.
Sun, G, Irvine KD.  2014.  Control of growth during regeneration.. Current topics in developmental biology. 108:95-120. AbstractWebsite
Regeneration is a process by which organisms replace damaged or amputated organs to restore normal body parts. Regeneration of many tissues or organs requires proliferation of stem cells or stem cell-like blastema cells. This regenerative growth is often initiated by cell death pathways induced by damage. The executors of regenerative growth are a group of growth-promoting signaling pathways, including JAK/STAT, EGFR, Hippo/YAP, and Wnt/β-catenin. These pathways are also essential to developmental growth, but in regeneration, they are activated in distinct ways and often at higher strengths, under the regulation by certain stress-responsive signaling pathways, including JNK signaling. Growth suppressors are important in termination of regeneration to prevent unlimited growth and also contribute to the loss of regenerative capacity in nonregenerative organs. Here, we review cellular and molecular growth regulation mechanisms induced by organ damage in several models with different regenerative capacities.
Luo, C, Dong J, Zhang Y, Lam E.  2014.  Decoding the role of chromatin architecture in development: coming closer to the end of the tunnel.. Frontiers in Plant Science. 5 AbstractWebsite
Form and function in biology are intimately related aspects that are often difficult to untangle. While the structural aspects of chromatin organization were apparent from early cytological observations long before the molecular details of chromatin functions were deciphered, the extent to which genome architecture may impact its output remains unclear. A major roadblock to resolve this issue is the divergent scales, both temporal and spatial, of the experimental approaches for examining these facets of chromatin biology. Recent advances in high-throughput sequencing and informatics to model and monitor genome-wide chromatin contact sites provide the much-needed platform to close this gap. This mini-review will focus on discussing recent efforts applying new technologies to elucidate the roles of genome architecture in coordinating global gene expression output. Our discussion will emphasize the potential roles of differential genome 3-D structure as a driver for cell fate specification of multicellular organisms. An integrated approach that combines multiple new methodologies may finally have the necessary temporal and spatial resolution to provide clarity on the roles of chromatin architecture during development.
Xu, A, Irvine KD.  2014.  Notch-ligand binding assays in Drosophila cells.. Methods in molecular biology (Clifton, NJ). 1187:277-284. AbstractWebsite
Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor-ligand interactions. We describe here a simple, widely used method for assaying receptor-ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity.
Mani, M, Goyal S, Irvine KD, Shraiman BI.  2013.  Collective polarization model for gradient sensing via Dachsous-Fat intercellular signaling.. Proceedings of the National Academy of Sciences of the United States of America. AbstractWebsite
Dachsous-Fat signaling via the Hippo pathway influences proliferation during Drosophila development, and some of its mammalian homologs are tumor suppressors, highlighting its role as a universal growth regulator. The Fat/Hippo pathway responds to morphogen gradients and influences the in-plane polarization of cells and orientation of divisions, linking growth with tissue patterning. Remarkably, the Fat pathway transduces a growth signal through the polarization of transmembrane complexes that responds to both morphogen level and gradient. Dissection of these complex phenotypes requires a quantitative model that provides a systematic characterization of the pathway. In the absence of detailed knowledge of molecular interactions, we take a phenomenological approach that considers a broad class of simple models, which are sufficiently constrained by observations to enable insight into possible mechanisms. We predict two modes of local/cooperative interactions among Fat-Dachsous complexes, which are necessary for the collective polarization of tissues and enhanced sensitivity to weak gradients. Collective polarization convolves level and gradient of input signals, reproducing known phenotypes while generating falsifiable predictions. Our construction of a simplified signal transduction map allows a generalization of the positional value model and emphasizes the important role intercellular interactions play in growth and patterning of tissues.