In Schizosaccharomyces pombe, systematic analyses of single transcription factor deletion or overexpression strains have made substantial advances in determining the biological roles and target genes of transcription factors, yet these characteristics are still relatively unknown for over a quarter of them. Moreover, the comprehensive list of proteins that regulate transcription factors remains incomplete. To further characterize Schizosaccharomyces pombe transcription factors, we performed synthetic sick/lethality and synthetic dosage lethality screens by synthetic genetic array. Examination of 2,672 transcription factor double deletion strains revealed a sick/lethality interaction frequency of 1.72%. Phenotypic analysis of these sick/lethality strains revealed potential cell cycle roles for several poorly characterized transcription factors, including SPBC56F2.05, SPCC320.03, and SPAC3C7.04. In addition, we examined synthetic dosage lethality interactions between 14 transcription factors and a miniarray of 279 deletion strains, observing a synthetic dosage lethality frequency of 4.99%, which consisted of known and novel transcription factor regulators. The miniarray contained deletions of genes that encode primarily posttranslational-modifying enzymes to identify putative upstream regulators of the transcription factor query strains. We discovered that ubiquitin ligase Ubr1 and its E2/E3-interacting protein, Mub1, degrade the glucose-responsive transcriptional repressor Scr1. Loss of ubr1(+) or mub1(+) increased Scr1 protein expression, which resulted in enhanced repression of flocculation through Scr1. The synthetic dosage lethality screen also captured interactions between Scr1 and 2 of its known repressors, Sds23 and Amk2, each affecting flocculation through Scr1 by influencing its nuclear localization. Our study demonstrates that sick/lethality and synthetic dosage lethality screens can be effective in uncovering novel functions and regulators of Schizosaccharomyces pombe transcription factors.

The competitive toxic and stress-inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA, and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study, we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of the effects of prolonged exposure to copper. In short, we recovered two copper homeostatic genes involved in transporting and assembling that are required in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit, and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched along with the deletion of several proteins involved in importing generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.

This study identifies a post‐transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA‐binding proteins in Schizosaccharomyces pombe . Cells expressing Puf2 and Puf4 stimulate decay of the frp1 + mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1 + mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron‐replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP‐ frp1 + 3′UTR RNAs was generated using a reporter gene containing the 3′ untranslated region (UTR) of frp1 + that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1 + 3′UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM‐homology RNA‐binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1 + 3′UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1 + mRNA under basal and iron‐replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.

•PUF genes are overrepresented in plant species.•Plant PUFs show extensive PUF repeat diversity.•Unique plant PUF subcellular localization patterns are apparent.•Functional roles for Arabidopsis PUF genes are emerging.•The RNA-binding domain of a nucleolar PUF has potential for engineering.

Oil sands surface mining generates vast quantities of oil sands process-affected water (OSPW) as a by-product of bitumen extraction. The acid extractable organic (AEO) fraction of OSPW contains several contaminants, including naphthenic acids (NAs). While responses of living organisms to NA and AEO exposure have been described at the developmental, physiological, metabolic and gene expression levels, the effects of these compounds at the cellular and subcellular level are limited. Using live cell fluorescence microscopy and a suite of fluorescent marker proteins, we studied the intracellular responses of the plant cell cytoskeleton and several membrane-bound organelles to NA and AEO treatments. A rapid disassembly of cortical microtubules and a decrease in dynamics associated with actin filaments was observed in response to these treatments. Concomitantly, the integrity and dynamics of mitochondria, peroxisomes, Golgi stacks, and endoplasmic reticulum were also altered. AEO treatments were the most toxic to cells and resulted in the accumulation reactive oxygen species. This study provides foundational evidence for intracellular responses to NA and AEO exposure using two evolutionarily diverse model plant cell types. This cellular assay could be used to identify the most toxic components of AEO sub-fractions, and assist in determining the effectiveness of OSPW remediation efforts.

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

Environmental contaminants are known to impair reproduction, metabolism and development in wild life and humans. To investigate the mechanisms underlying adverse effects of contaminants, fathead minnows were exposed to a number of endocrine disruptive chemicals (EDCs) including Nonylphenol (NP), bisphenol-A (BPA), Di(2-ethylhexyl) phthalate (DEHP), and a mixture of the three chemicals for 21 days, followed by determination of the liver transcriptome by expression microarrays. Pathway analysis revealed a distinct mode of action for the individual chemicals and their mixture. The results showed expression changes in over 980 genes in response to exposure to these EDC contaminants individually and in mixture. Ingenuity Pathway core and toxicity analysis were used to identify the biological processes, pathways and the top regulators affected by these compounds. A number of canonical pathways were significantly altered, including cell cycle & proliferation, lipid metabolism, inflammatory, innate immune response, stress response, and drug metabolism. We identified 18 genes that were expressed in all individual and mixed treatments. Relevant candidate genes identified from expression microarray data were verified using quantitative PCR. We were also able to identify specific genes affected by NP, BPA, and DEHP individually, but were also affected by exposure to the mixture of the contaminants. Overall the results of this study provide novel information on the adverse health impact of contaminants tested based on pathway analysis of transcriptome data. Furthermore, the results identify a number of new biomarkers that can potentially be used for screening environmental contaminants.

It is essential to understand the mechanisms by which a toxicant is capable of poisoning the bacterial cell. The mechanism of action of many biocides and toxins, including numerous ubiquitous compounds, is not fully understood. For example, despite the widespread clinical and commercial use of silver (Ag), the mechanisms describing how this metal poisons bacterial cells remains incomplete. To advance our understanding surrounding the antimicrobial action of Ag, we performed a chemical genetic screen of a mutant library of Escherichia coli—the Keio collection, in order to identify Ag sensitive or resistant deletion strains. Indeed, our findings corroborate many previously established mechanisms that describe the antibacterial effects of Ag, such as the disruption of iron-sulfur clusters containing proteins and certain cellular redox enzymes. However, the data presented here demonstrates that the activity of Ag within the bacterial cell is more extensive, encompassing genes involved in cell wall maintenance, quinone metabolism and sulfur assimilation. Altogether, this study provides further insight into the antimicrobial mechanism of Ag and the physiological adaption of E. coli to this metal.

Some early studies suggested that post-operative thoracic radiotherapy (PORT) using non-modern radiation techniques in non-small cell lung cancer (NSCLC) might cause significant treatment-related toxicities. Higher radiation doses to the heart have been recently linked to more cardiac events and worse overall survival (OS) in patients with locally-advanced NSCLC treated with thoracic irradiation. We embarked on a national population-based study to assess the association between radiation heart doses, acute myocardial infarct (AMI) rates and OS in NSCLC patients treated with PORT using contemporary radiation techniques. Study eligibility criteria included stage I to III NSCLC treated with PORT from 2007 to 2014 in our public hospitals which comprised about 80% of national caseload. The clinical and dosimetric data were collected from the institutional electronic medical records and linked to the national death and AMI registries. Univariate Cox regression was performed using STATA version 13. 43 eligible patients were identified. Median follow-up duration was 36.6 months. Characteristics of study population are summarized in Table 1. There were no AMI events. The 1- and 2-year OS were 74% and 65%. Univariate Cox regression analysis showed that age (hazard ratio, 1.06; 95% confidence interval, 1.01 to 1.10; P= 0.008) was the only significant factor associated with OS. Radiation heart doses, including mean heart dose, volume of heart receiving at least 5, 25, 30, 40, 50 Gy and dose to 30% of heart volume, were not associated with OS. This study found that various radiation heart doses were not significantly associated with OS in patients with NSCLC treated with PORT. Studies with larger sample size and longer term follow-up are needed to assess cardiac outcome, given the possibility of late occurrence of AMI events.

BackgroundSilver is being widely‐deployed in the clinic – in wound dressing, catheters, and endotracheal tubes ‐ to combat and control infectious disease. Despite this widespread use, we still don't know the precise way that silver kills the bacterial cell. Recently, our research group demonstrated that silver formulations with novel chemistries – silver oxysalts – have an enhanced antibacterial activity. Yet, we still don't know why different silver formulations show variable antimicrobial efficacy.MethodsTo test the efficacy of various silver compounds, numerous strains of E.coli, P.aeruginosa, and S.aureus were grown as single and multispecies biofilms in the Calgary Biofilm Device. The capacity of different silver compounds to prevent the formation of and eradicate planktonic and biofilm populations of bacteria was performed using the minimal biofilm eradication concentration assay, confocal microscopy and crystal violet staining. To enhance our understanding of how silver poisons bacteria, we undertook a robotic chemical genetic screen of an ordered mutant library of E.coli bacteria – the Keio collection. To confirm the genetic linkage of our silver responsive genes identified in our chemical genetic screen, we used Scarless Cas‐9 Assisted Recombineering to generate unmarked mutants. Our genes of interest were then linked to silver sensitivity or resistance phenotypes using minimal inhibition concentration assays and transmission electron microscopy (TEM).ResultsWe observed that higher oxidation states of silver have enhanced activity for preventing the formation of, and eradicating single and multispecies, planktonic and biofilm, populations of bacteria – suggesting that the chemistry of silver formulations dictates its antimicrobial efficacy. Our chemical genetic screen suggests that silver poisoning has previously unanticipated effects on the bacterial cell including disrupting the bacterial cell envelope, altering indole metabolism, and controlling bacterial cell population. Moreover, using a Recombineering workflow and TEM we confirmed the involvement of a gene involved in the production of a cell wall protein, and a monovalent cation transporter, in silver sensitivity and resistance, respectively. The latter finding is relevant because the protein responsible for the entrance of silver into the bacterial cell has yet to be identified.ConclusionsAltogether, our findings established novel mechanisms regarding the mode‐of‐action of silver in the bacterial cell. We've established that the specific chemistries of silver compounds determine antimicrobial capacity. Additionally, we've identified genes that may lead to silver resistance. These findings are ever‐important as we aim to maintain the utility of this valuable antimicrobial agent.Support or Funding InformationWe graciously acknowledge funding from the Natural Science and Engineering Research Council of Canada and the Canadian Institutes of Health Research. JL was funded by a Banting Postdoctoral Fellowship and an Alberta Innovates Health Solutions Postdoctoral award. We would also like to thank the University of Calgary for providing an Eyes High Graduate/Postgraduate Fellowship to NG and HA, respectively and research funding for JL.

Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1+ increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network.

Naphthenic acids (NAs), a class of structurally diverse carboxylic acids with often complex ring structures and large aliphatic tail groups, are important by-products of many petrochemical processes including the oil sands mining activity of Northern Alberta. While it is evident that NAs have both acute and chronic harmful effects on many organisms, many aspects of their toxicity remain to be clarified. Particularly, while substantive data sets have been collected on NA toxicity in aquatic prokaryote and vertebrate model systems, to date, nothing is known about the toxic effects of these compounds on the embryonic development of aquatic invertebrate taxa, including freshwater mollusks. This study examines under laboratory conditions the toxicity of NAs extracted from oil sands process water (OSPW) and the low-molecular weight model NAs cyclohexylsuccinic acid (CHSA), cyclohexanebutyric acid (CHBA), and 4-tert-butylcyclohexane carboxylic acid (4-TBCA) on embryonic development of the snail Lymnaea stagnalis, a common freshwater gastropod with a broad Palearctic distribution. Evidence is provided for concentration-dependent teratogenic effects of both OSPW-derived and model NAs with remarkably similar nominal threshold concentrations between 15 and 20 mg/L and 28d EC50 of 31 mg/L. In addition, the data provide evidence for substantial toxicokinetic differences between CHSA, CHBA and 4-TBCA. Together, our study introduces Lymnaea stagnalis embryonic development as an effective model to assay NA-toxicity and identifies molecular architecture as a potentially important toxicokinetic parameter in the toxicity of low-molecular weight NA in embryonic development of aquatic gastropods.

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.

Introduction The emergence of the post‐antibiotic era has left us desperate for alternative strategies to combat infectious disease. Historically, silver has been used to prevent and control microbial infections. Silver is now seeing widespread use in a variety of medical devices including bandages, catheters, and endotracheal tubes; as well as numerous household items. Yet, we still don't mechanistically understand how silver poisons the microbial cell. Objectives Recently, our research group demonstrated that the higher oxidation states of silver have an enhanced capacity to eradicate laboratory and antibiotic‐resistant strains of bacteria – both planktonic bacteria as well as biofilms. Our current research objective is to understand the mechanistic details of how silver exerts its toxicity on the bacterial cell. Methodology To address the mechanisms of how silver kills bacterial cells, we used a high‐throughput, toxicogenomic approach ‐ screening a knockout collection of E.coli K12 (the Keio collection) for Ag‐sensitive/resistant mutants. Following our toxicogenomic screen, markerless deletion mutants were generated from statistically significant hits using the Red Recombinase system. The markerless genetic mutants were then subjected to phenotypic and biochemical assays to verify their resistance or sensitivity profile to silver treatment. Results Although silver is thought to exert its toxicity by disrupting thiol metabolism and cellular redox‐status, our results demonstrate that silver poisons E.coli through a variety of previously unestablished mechanisms. These include interfering with genes involved in maintaining cell wall integrity, toxin/antitoxin systems, nutrient catabolism, and quorum sensing. Conclusion Here we demonstrate how a toxicogenomic approach was used to determine the mechanistic manner by which silver poisons the bacterial cell. To that end, the robust nature of this methodology can be used to screen other metals, toxins, and antimicrobials to determine their mechanisms of action. Mechanisms that are desperately needed to develop novel antimicrobial strategies. Support or Funding Information This work has been supported by the Natural Science and Engineering Council of Canada. J.L. is supported by a Banting Postdoctoral Fellowship and an Alberta Innovates Health Solutions Postdoctoral Fellowship Award.

The ability to mitigate toxicity of oil sands process-affected water (OSPW) for return into the environment is an important issue for effective tailings management in Alberta, Canada. OSPW toxicity has been linked to classical naphthenic acids (NAs), but the toxic contribution of other acid-extractable organics (AEOs) remains unknown. Here, we examine the potential for in situ bioremediation of OSPW AEOs by indigenous algae. Phosphate biostimulation was performed in OSPW to promote the growth of indigenous photosynthetic microorganisms and subsequent toxicity and chemical changes were determined. After 12 weeks, the AEO fraction of phosphate-biostimulated OSPW was significantly less toxic to the fission yeast Schizosaccharomyces pombe than unstimulated OSPW. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) analysis of the AEO fraction in phosphate-biostimulated OSPW showed decreased levels of SO3 class compounds, including a subset that may represent linear arylsulfonates. A screen with S. pombe transcription factor mutant strains for growth sensitivity to the AEO fraction or sodium dodecylbenzenesulfonate revealed a mode of toxic action consistent with oxidative stress and detrimental effects on cellular membranes. These findings demonstrate a potential algal-based in situ bioremediation strategy for OSPW AEOs and uncover a link between toxicity and AEOs other than classical NAs.

Bitumen mining in the Athabasca oil sands region of northern Alberta results in the accumulation of large volumes of oil sands process-affected water (OSPW). The acid-extractable organic (AEO) fraction of OSPW contains a variety of compounds, including naphthenic acids, aromatics, and sulfur- and nitrogen-containing compounds that are toxic to aquatic and terrestrial organisms. We have studied the effect of AEO treatment on the transcriptome of root and shoot tissues in seedlings of the model plant, Arabidopsis thaliana. Several genes encoding enzymes involved in the xenobiotic detoxification pathway were upregulated, including cytochrome P450s (CYPs), UDP-dependent glycosyltransferases (UGTs), glutathione-S-transferases (GSTs), and membrane transporters. In addition, gene products involved in oxidative stress, β-oxidation, and glucosinolate degradation were also upregulated, indicating other potential mechanisms of the adaptive response to AEO exposure. These results provide insight into the pathways that plants use to detoxify the organic acid component of OSPW. Moreover, this study advances our understanding of genes that could be exploited to potentially develop phytoremediation and biosensing strategies for AEO contaminants resulting from oil sands mining.

Lesions in DNA can block replication fork progression, leading to its collapse and gross chromosomal rearrangements. To circumvent such outcomes, the DNA damage tolerance (DDT) pathway becomes engaged, allowing the replisome to bypass a lesion and complete S phase. Chromatin remodeling complexes have been implicated in the DDT pathways, and here we identify the NuA4 remodeler, which is a histone acetyltransferase, to function on the translesion synthesis (TLS) branch of DDT. Genetic analyses in Saccharomyces cerevisiae showed synergistic sensitivity to MMS when NuA4 alleles, esa1-L254P and yng2Δ, were combined with the error-free bypass mutant ubc13Δ. The loss of viability was less pronounced when NuA4 complex mutants were disrupted in combination with error-prone/TLS factors, such as rev3Δ, suggesting an epistatic relationship between NuA4 and error-prone bypass. Consistent with cellular viability measurements, replication profiles after exposure to MMS indicated that small regions of unreplicated DNA or damage were present to a greater extent in esa1-L254P/ubc13Δ mutants, which persist beyond the completion of bulk replication compared to esa1-L254P/rev3Δ. The critical role of NuA4 in error-prone bypass is functional even after the bulk of replication is complete. Underscoring this observation, when Yng2 expression is restricted specifically to G2/M of the cell cycle, viability and TLS-dependent mutagenesis rates were restored. Lastly, disruption of HTZ1, which is a target of NuA4, also resulted in mutagenic rates of reversion on level with esa1-L254P and yng2Δ mutants, indicating that the histone variant H2A.Z functions in vivo on the TLS branch of DDT.

Dear Editor, Self-incompatibility (SI) is a genetic mechanism through which flowering plants prevent self-pollination to ensure out-crossing and genetic diversity. In Brassica sp., this mechanism is controlled by the self-incompatibility (S) locus, in which, the stigmatic 'S-locus receptor kinase (SRK)' recognizes the 'S-locus cysteine rich protein (SCR)' from the self-pollen to elicit an active rejection response. This results in blocking of compatibility factors from being delivered to the site of pollen attachment leading to self-pollen rejection (Chapman and Goring, 2010Chapman L.A. Goring D.R Pollen–pistil interactions regulating successful fertilization in the Brassicaceae.J. Exp. Bot. 2010; 61: 1987-1999Crossref PubMed Scopus (92) Google Scholar). In contrast, following recognition of compatible signals from the cross-pollen or compatible pollen (CP), the stigma releases its resources such as water and nutrients to the dry pollen so that the pollen tube can germinate and penetrate the stigmatic cuticle leading to successful fertilization. Thus, an incompatible or self-pollen is fully capable of eliciting a compatible response, but is actively rejected before compatible responses can occur. Following landing of self-pollen or cross-pollen on stigmas of Brassica napus (canola), there is a latent period of 30min when signals are exchanged between the highly lipophilic pollen coat proteins and the stigmatic components. CP tubes can be observed to emerge between 30 and 90min after initiation of this interaction. Given that stigmas control the outcome of pollen acceptance or rejection, deciphering the transcriptional changes during this latent period would reveal genes involved in compatible and self-incompatible responses. As expected, when self-incompatible W1 canola stigmas were stained with aniline blue to observe pollen tubes, SI-pollinated stigmas lacked any pollen attachment or pollen tubes at 30min and 6h after pollination (Figure 1A, right panel). The weakly attached pollen without any positive interactions is washed away during the staining process. Following compatible pollination, although pollen attachment and pollen tubes could be observed at 6h, at 30min after pollination, no pollen attachment could be observed (Figure 1A, right panel). This is due to lack of complete adhesion and pollen tube germination at 30min after pollination. These observations suggest that analyzing the transcriptome changes 0–30min following SI and compatible pollinations would likely reveal genes that are triggered by pollen landing on the stigma and could represent genes that are required for promoting SI and compatible responses, respectively. To identify the genes that are differentially regulated by SI and compatible pollinations (Figure 1A, left panel), RNA extracted from self-incompatible W1 stigmas, pollinated with self-pollen or cross-pollen for either 15 or 30min, were compared against RNA from unpollinated (UP) stigmas through transcriptome profiling, using the Agilent 4×44K Brassica Gene Expression Microarrays (G2519F). Following normalization, filtering based on P-values (<0.001) and then by two-fold up-regulation in at least one of the microarray experiments, we identified 621 genes that were differentially regulated. Clustering of these genes (Supplemental Table 1) clearly indicated strong up-regulation of multiple genes across all four treatments. This suggested to us that these were likely expressed pollen genes when SI and compatible pollinated stigmas were compared with UP stigmas that lacked any pollen. Utilizing the high sequence similarity between Arabidopsis and Brassica, we identified the orthologous Arabidopsis genes for the 621 canola genes to facilitate further bioinformatic analyses. Following filtering of the pollen genes from the differentially expressed genes in the microarray experiments (see Supplementary Data), the 621 genes were subdivided into stigma genes (287), pollen genes (181), and stigma–pollen genes (153) (Supplemental Table 2). Since the focus of this study was to identify stigmatic genes specifically regulated by SI and CP, we focused our attention on the 287 stigmatic genes. Clustering the stigmatic genes (Supplemental Table 3) revealed a clear pattern of changes between SI and compatible pollination. Heat maps were generated for the subset of genes that were specifically induced or repressed in SI and compatible pollination (Figure 1B–1E and Supplemental Table 4). There were only three genes that displayed specific up-regulation following SI pollination (Figure 1B). These included AT3G43220/SAC3, a phosphoinositide phosphatase; AT4G33990, a pentatricopeptide repeat-containing protein; and AT3G03740, a BTB/POZ domain-containing protein that serves as a protein adaptor for cullin-based E3 ligase-mediated proteasomal degradation in plants. The major pathway through which SI functions is through ARC1 (U-box E3 ligase)-mediated proteasomal degradation of compatibility factors leading to pollen rejection (Samuel et al., 2008Samuel M.A. Yee D. Haasen K.E. Goring D.R 'Self' pollen rejection through the intersection of two cellular pathways in the Brassicaceae: self-incompatibility and the compatible pollen response.in: Franklin-Tong V.E Self-Incompatibility in Flowering Plants: Evolution, Diversity, and Mechanisms. Springer-Verlag, Berlin2008: 175-191Crossref Scopus (20) Google Scholar). Rapid induction of AT3G03740 would suggest that, once SI is activated through ARC1, the AT3G03740 could function to enhance the proteasomal degradation pathway to synergistically influence the SI response. The lack of up-regulation of many SI-specific genes following SI pollination is quite consistent with the proteomics study that reported only down-regulation of multiple proteins following SI without any differential up-regulation (Samuel et al., 2011Samuel M.A. Tang W. Jamshed M. Northey J. Patel D. Smith D. Siu K.W. Muench D.G. Wang Z.Y. Goring D.R Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during pollen responses.Mol. Cell Proteomics. 2011; 10 (M111 011338)Crossref Scopus (52) Google Scholar). This observation further strengthens the hypothesis that most of the SI-specific events are posttranslational events so that the rejection response occurs at a faster pace than the compatible response. Out of the 12 genes that were specifically repressed following SI pollination (Figure 1C), AT1G72290, a Kunitz domain-containing protease (trypsin) inhibitor, was particularly interesting, since it showed rapid and consistent down-regulation at 15 and 30min following SI pollination. Protease inhibitors are small proteins that interact with proteases and maintain the fully active proteases in a partially active or inactive state that can be activated by release of these inhibitors (Habib and Fazili, 2007Habib H. Fazili K.M Plant protease inhibitors: a defense strategy in plants.Biotechnol. Mol. Biol. Rev. 2007; 2: 68-85Google Scholar). Following SI, a sudden burst of proteasomal activity and ubiquitination is known to occur (Stone et al., 2003Stone S.L. Anderson E.M. Mullen R.T. Goring D.R ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen.Plant Cell. 2003; 15: 885-898Crossref PubMed Scopus (271) Google Scholar). The Kunitz trypsin inhibitor could potentially maintain the proteasomal proteases at a low active state prior to pollination and, following self-pollen attachment, this inhibition could be relieved to potentiate a rapid induction of proteasomal activity. Following compatible pollination, 14 genes were specifically down-regulated (Figure 1D). These included transcription factors (TF) AT3G24850, a DUF domain TF and MYB56 (AT5G17800), cell wall modifying enzyme beta-xylosidase (AT5G49360 (BXL1)) and AT1G05830 (ATX2), a histone–lysine N-methyltransferase that was down-regulated more than four-fold at 30min following compatible pollination. Reduction of ATX2 could release the transcriptional inhibition imposed by the tightly wound chromatin resulting in a sudden burst of transcriptional activity following compatible pollination. A number of genes (29) including MYB44 (AT5G67300), MYB77 (AT3G50060), and citric acid cycle-associated genes were strongly up-regulated following compatible pollination (Figure 1E and Supplemental Figure 1), indicating a system preparing itself for pollen tube penetration. When these 29 genes were subjected to gene ontology analysis using the Princeton GO Term Finder (http://go.princeton.edu/cgi-bin/GOTermFinder ), these genes were enriched in genes for lipid transport (17.2%, p-value = 0.0002). The representation of genes involved in lipid-transfer and lipid binding (AT5G59310, AT5G59320, AT5G59330, and AT3G22600) is quite consistent with previous observations that have reported a role for long-chain lipids in pollen hydration. The identities of the genes involved in this process of adding lipids to the cuticular surface of the papillary cells, to form a hydraulic conduit for transport of water to the pollen, have remained unknown (Dickinson, 1995Dickinson H Dry stigmas, water and self-incompatibility in Brassica.Sexual Plant Reproduction. 1995; 8: 1-10Crossref Scopus (161) Google Scholar; Samuel et al., 2008Samuel M.A. Yee D. Haasen K.E. Goring D.R 'Self' pollen rejection through the intersection of two cellular pathways in the Brassicaceae: self-incompatibility and the compatible pollen response.in: Franklin-Tong V.E Self-Incompatibility in Flowering Plants: Evolution, Diversity, and Mechanisms. Springer-Verlag, Berlin2008: 175-191Crossref Scopus (20) Google Scholar). The identification of multiple genes involved in phosphoinositol metabolism or transport sheds light on a possible early role for phosphatidylinositol phosphates (PtInsPS) signaling, which have been previously implicated to have a stigmatic role in supporting pollen hydration (Chapman and Goring, 2011Chapman L. Goring D.R Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility.Sex. Plant Reprod. 2011; 24: 319-326Crossref PubMed Scopus (12) Google Scholar). The inositol transporter, INT1 (AT2G43330) (Supplemental Figure 1), and the plasma membrane-localized PcaP1 (AT4G20260), proposed to be involved in release of PtInsPS (Kato et al., 2010Kato M. Nagasaki-Takeuchi N. Ide Y. Tomioka R. Maeshima M PCaPs, possible regulators of PtdInsP signals on plasma membrane.Plant Signaling & Behavior. 2010; 5: 848-850Crossref PubMed Scopus (22) Google Scholar), were up-regulated by compatible pollination. Interestingly, following SI pollination, induction of PtIns-phosphatase (AT3G43220) was observed, suggesting that SI could likely negate the generation of the PtInsPS (IP3) required to induce calcium influx essential for compatible pollinations (Iwano et al., 2004Iwano M. Shiba H. Miwa T. Che F.S. Takayama S. Nagai T. Miyawaki A. Isogai A Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis.Plant Physiol. 2004; 136: 3562-3571Crossref PubMed Scopus (131) Google Scholar). Several senescence-associated genes were up-regulated following compatible pollination (Figure 1F and Supplemental Table 5). Senescence-associated SAG12 and SAG29, lipid-transfer proteins (LTP3 and LTP4), ethylene-responsive TF (ERF012, ERF017, and ERF109), BCL-2 athanogene 6 (BAG6), and inositol transporter 1 (INT1) have been shown to be induced under senescence responses (Gepstein et al., 2003Gepstein S. Sabehi G. Carp M.J. Hajouj T. Nesher M.F. Yariv I. Dor C. Bassani M Large-scale identification of leaf senescence-associated genes.Plant J. 2003; 36: 629-642Crossref PubMed Scopus (319) Google Scholar). These genes were either down-regulated (SAG29, LTP3, 4, and 6) or had no noticeable differential expression after self-incompatible pollination, as evidenced by their expression in the transcriptome analysis and validation through qRT–PCR (Figure 1F and 1G). To examine whether compatible pollination induced a rapid senescence process in the papillary cells, we stained SI and compatible pollinated stigmas at various times after pollination with propidium iodide, followed by fluorescence microscopy. The observations indicated that compatible pollination induced a rapid senescence process with >40% papillary cells showing cell death at 6h and >70% at 24h, while UP and SI-pollinated stigmas displayed <40% and <30% dead cells, respectively, at 24h (Figure 1H and 1I). Lack of cell death following SI pollination indicates that the stigma, despite being pollinated, prevents the senescence programming from occurring to stay receptive for the right mate. Thus, our study has revealed that, within 30min after landing on the stigmatic surface, self-pollen and cross-pollen trigger a unique transcriptional program that would culminate in either rejection or acceptance of the pollen, respectively. Identification of these genes unveils a number of previously unknown mechanisms through which pollination can be controlled by the stigmas. Supplementary Data are available at Molecular Plant Online. This work was supported by Natural Sciences and Engineering Research Council of Canada grants and start-up funds from University of Calgary to M.A.S. and G.C. No conflict of interest declared.

Mapping transcriptional-regulatory networks requires the identification of target genes, binding specificities and signalling pathways of transcription factors. However, the characterization of each transcription factor sufficiently for deciphering such networks remains laborious. The recent availability of overexpression and deletion strains for almost all of the transcription factor genes in the fission yeast Schizosaccharomyces pombe provides a valuable resource to better investigate transcription factors using systematic genetics. In the present paper, I review and discuss the utility of these strain collections combined with transcriptome profiling and genome-wide chromatin immunoprecipitation to identify the target genes of transcription factors.