Category: Publications

Bassel GW, Lan H, Glaab E, Gibbs DJ, Gerjets T, Krasnogor N, Bonner AJ, Holdsworth MJ, Provart NJ

Proc. Natl. Acad. Sci. U.S.A. 2011 Jun;108(23):9709-14

PubMed PMID: 21593420

Abstract

Seed germination is a complex trait of key ecological and agronomic significance. Few genetic factors regulating germination have been identified, and the means by which their concerted action controls this developmental process remains largely unknown. Using publicly available gene expression data from Arabidopsis thaliana, we generated a condition-dependent network model of global transcriptional interactions (SeedNet) that shows evidence of evolutionary conservation in flowering plants. The topology of the SeedNet graph reflects the biological process, including two state-dependent sets of interactions associated with dormancy or germination. SeedNet highlights interactions between known regulators of this process and predicts the germination-associated function of uncharacterized hub nodes connected to them with 50% accuracy. An intermediate transition region between the dormancy and germination subdomains is enriched with genes involved in cellular phase transitions. The phase transition regulators SERRATE and EARLY FLOWERING IN SHORT DAYS from this region affect seed germination, indicating that conserved mechanisms control transitions in cell identity in plants. The SeedNet dormancy region is strongly associated with vegetative abiotic stress response genes. These data suggest that seed dormancy, an adaptive trait that arose evolutionarily late, evolved by coopting existing genetic pathways regulating cellular phase transition and abiotic stress. SeedNet is available as a community resource (http://vseed.nottingham.ac.uk) to aid dissection of this complex trait and gene function in diverse processes.

O’Brien HE, Thakur S, Guttman DS

Annu Rev Phytopathol 2011;49:269-89

PubMed PMID: 21568703

Abstract

The phytopathogenic bacterium Pseudomonas syringae causes serious diseases in a wide range of important crop plants, with recent severe outbreaks on the New Zealand kiwifruit crop and among British horse chestnut trees. Next-generation genome sequencing of over 25 new strains has greatly broadened our understanding of how this species adapts to a diverse range of plant hosts. Not unexpectedly, the genomes were found to be highly dynamic, and extensive polymorphism was found in the distribution of type III secreted effectors (T3SEs) and other virulence-associated genes, even among strains within the same pathovar. An underexplored area brought to light by these data is the specific metabolic adaptations required for growth on woody hosts. These studies provide a tremendous wealth of candidates for more refined functional characterization, which is greatly enhancing our ability to disentangle the web of host-pathogen interactions that determine disease outcomes.

Fucile G, Garcia C, Carlsson J, Sunnerhagen M, Christendat D

Protein Sci. 2011 Jul;20(7):1125-36

PubMed PMID: 21520319

Abstract

The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 °C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.

Austin RS, Vidaurre D, Stamatiou G, Breit R, Provart NJ, Bonetta D, Zhang J, Fung P, Gong Y, Wang PW, McCourt P, Guttman DS

Plant J. 2011 Aug;67(4):715-25

PubMed PMID: 21518053

Abstract

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F? population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F? population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F? lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

Winter CM, Austin RS, Blanvillain-Baufumé S, Reback MA, Monniaux M, Wu MF, Sang Y, Yamaguchi A, Yamaguchi N, Parker JE, Parcy F, Jensen ST, Li H, Wagner D

Dev. Cell 2011 Apr;20(4):430-43

PubMed PMID: 21497757

Abstract

The transition from vegetative growth to flower formation is critical for the survival of flowering plants. The plant-specific transcription factor LEAFY (LFY) has central, evolutionarily conserved roles in this process, both in the formation of the first flower and later in floral patterning. We performed genome-wide binding and expression studies to elucidate the molecular mechanisms by which LFY executes these roles. Our study reveals that LFY directs an elaborate regulatory network in control of floral homeotic gene expression. LFY also controls the expression of genes that regulate the response to external stimuli in Arabidopsis. Thus, our findings support a key role for LFY in the coordination of reproductive stage development and disease response programs in plants that may ensure optimal allocation of plant resources for reproductive fitness. Finally, motif analyses reveal a possible mechanism for stage-specific LFY recruitment and suggest a role for LFY in overcoming polycomb repression.

Murray RL, Kozlowska JL, Cutter AD

BMC Evol. Biol. 2011;11:99

PubMed PMID: 21492473

Abstract

BACKGROUND: Sperm competition is a driving force in the evolution of male sperm characteristics in many species. In the nematode Caenorhabditis elegans, larger male sperm evolve under experimentally increased sperm competition and larger male sperm outcompete smaller hermaphrodite sperm for fertilization within the hermaphrodite reproductive tract. To further elucidate the relative importance of sperm-related traits that contribute to differential reproductive success among males, we quantified within- and among-strain variation in sperm traits (size, rate of production, number transferred, competitive ability) for seven male genetic backgrounds known previously to differ with respect to some sperm traits. We also quantified male mating ability in assays for rates of courtship and successful copulation, and then assessed the roles of these pre- and post-mating traits in first- and second-male fertilization success.

RESULTS: We document significant variation in courtship ability, mating ability, sperm size and sperm production rate. Sperm size and production rate were strong indicators of early fertilization success for males that mated second, but male genetic backgrounds conferring faster sperm production make smaller sperm, despite virgin males of all genetic backgrounds transferring indistinguishable numbers of sperm to mating partners.

CONCLUSIONS: We have demonstrated that sperm size and the rate of sperm production represent dominant factors in determining male fertilization success and that C. elegans harbors substantial heritable variation for traits contributing to male reproductive success. C. elegans provides a powerful, tractable system for studying sexual selection and for dissecting the genetic basis and evolution of reproduction-related traits.

Sharifpoor S, Nguyen Ba AN, Youn JY, Young JY, van Dyk D, Friesen H, Douglas AC, Kurat CF, Chong YT, Founk K, Moses AM, Andrews BJ

Genome Biol. 2011;12(4):R39

PubMed PMID: 21492431

Abstract

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future.

Souza BR, Romano-Silva MA, Tropepe V

J. Neurosci. 2011 Apr;31(14):5512-25

PubMed PMID: 21471388

Abstract

An imbalance in dopamine-mediated neurotransmission is a hallmark physiological feature of neuropsychiatric disorders, such as schizophrenia. Recent evidence demonstrates that dopamine D(2) receptors, which are the main target of antipsychotics, modulate the activity of the protein kinase Akt, which is known to be downregulated in the brain of patients with schizophrenia. Akt has an important role in the regulation of cellular processes that are critical for neurodevelopment, including gene transcription, cell proliferation, and neuronal migration. Thus, it is possible that during brain development, altered Akt-dependent dopamine signaling itself may lead to defects in neural circuit formation. Here, we used a zebrafish model to assess the direct impact of altered dopamine signaling on brain development and larval motor behavior. We demonstrate that D(2) receptor activation acutely suppresses Akt activity by decreasing the level of pAkt(Thr308) in the larval zebrafish brain. This D(2)-dependent reduction in Akt activity negatively regulates larval movement and is distinct from a D(1)-dependent pathway with opposing affects on motor behavior. In addition, we show that D(2)-dependent suppression of Akt activity causes a late onset change in GSK3b activity, a known downstream target of Akt signaling. Finally, altered D(2) receptor signaling, or direct inhibition of Akt activity, causes a significant decrease in the size of the GABAergic neuron population throughout most of the brain. Our observations suggest that D(2) receptor signaling suppresses Akt-GSK3b activity, which regulates GABAergic neuron development and motor behavior.

Sanghera KP, Mathalone N, Baigi R, Panov E, Wang D, Zhao X, Hsu H, Wang H, Tropepe V, Ward M, Boyd SR

Mol. Cell. Neurosci. 2011 Jun;47(2):145-53

PubMed PMID: 21463685

Abstract

Oxygen (O?) tension has emerged as a major regulator of stem cell (SC) biology. Low O? concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H?O?) induced damage and resistant to moderate levels of low oxygen stress (1% O?). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O?) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight.

Moeder W, Urquhart W, Ung H, Yoshioka K

Mol Plant 2011 May;4(3):442-52

PubMed PMID: 21459831

Abstract

Since the first plant cyclic nucleotide-gated ion channel (CNGC), HvCBT1, was identified as a calmodulin binding protein, more than a decade has passed and a substantial amount of work has been done to understand the molecular nature and function of these channel proteins. Based on electrophysiological and heterologous expression analyses, plant CNGCs function as non-selective cation channels and, so far, their biological roles have been reported in defense responses, development, and ion homeostasis. Forward genetic approaches identified four AtCNGCs (AtCNGC2, 4, 11, and 12) to be involved in plant immunity, as null mutants for AtCNGC2, 4, 11, and 12 as well as a gain-of- function mutant for AtCNGC11 and 12 exhibited alterations in defense responses. Since ion flux changes have been reported as one of the early events upon pathogen recognition and also are an essential component for the activation of defense responses, the involvement of CNGCs in these ion flux changes has been suggested. However, the recent detailed characterization of null mutants suggested a more complex involvement of this channel family. In this review, we focus on the discoveries and characterization of these CNGC mutants and discuss possible roles of CNGCs as components in plant immunity.