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Michael Deyholos

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Michael Deyholos

Department of Biological Sciences

deyholos{at}ualberta{dot}ca

  • Plant Biotechnology
  • Genetics
  • Molecular Biology
  • Genomics

  • BIOL 207
  • BOT 382
  • PLSCI 491

http://hdl.handle.net/10402/era.22934

Subject areas and related deposits

  • Celled cotton fiber

    • A flax fibre proteome: identification of proteins enriched in bast fibres

      Background: Bast fibres from the phloem tissues of flax are scientifically interesting and economically useful due in part to a dynamic system of secondary cell wall deposition. To better understand the molecular mechanisms underlying the process of cell wall development in flax, we extracted proteins from individually dissected phloem fibres (i.e. individual cells) at an early stage of secondary cell wall development, and compared these extracts to protein extracts from surrounding, non-fibre cells of the cortex, using fluorescent (DiGE) labels and 2D-gel electrophoresis, with identities assigned to some proteins by mass spectrometry. Results: The abundance of many proteins in fibres was notably different from the surrounding non-fibre cells of the cortex, with approximately 13% of the 1,850 detectable spots being significantly (> 1.5 fold, p ≤ 0.05) enriched in fibres. Following mass spectrometry, we assigned identity to 114 spots, of which 51 were significantly enriched in fibres. We observed that a K+ channel subunit, annexins, porins, secretory pathway components, β-amylase, β-galactosidase and pectin and galactan biosynthetic enzymes were among the most highly enriched proteins detected in developing flax fibres, with many of these proteins showing electrophoretic patterns consistent with post-translational modifications. Conclusion: The fibre-enriched proteins we identified are consistent with the dynamic process of secondary wall deposition previously suggested by histological and biochemical analyses, and particularly the importance of galactans and the secretory pathway in this process. The apparent abundance of β-amylase suggests that starch may be an unappreciated source of materials for cell wall biogenesis in flax bast fibres. Furthermore, our observations confirm previous reports that correlate accumulation proteins such as annexins, and specific heat shock proteins with secondary cell wall deposition.

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  • Floral Homeotic Gene

    • Separable whorl-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron

      We analyzed the 4-kb intragenic control region of the AGAMOUS (AG) gene to gain insight into the mechanisms controlling its expression during early flower development. We identified three major expression patterns conferred by 19 AG::reporter gene constructs: the normal AG pattern, a stamen-specific pattern, and a predominantly carpel pattern. To determine whether these three expression patterns were under negative control by APETALA2 (AP2) or LEUNIG (LUG), we analyzed b-glucuronidase staining patterns in Arabidopsis plants homozygous for strong ap2 and lug mutations. Our results indicated that the stamen-specific pattern was independent of AP2 but dependent on LUG; conversely, the carpel-specific pattern was independent of LUG but dependent on AP2. These results lead to a model of control of AG expression such that expression in each of the two inner whorls is under independent positive and negative control.

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  • Gene expression

    • The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

      Background: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions: The technique detailed here minimizes the cost and effort to replicate a PCRgenerated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.

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  • Genetics

  • Genetics, molecular biology, undergraduate teaching, textbook

  • Length CDNA microarray

    • Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

      Background: Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks. Results: We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or downregulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like); signalling molecules (e.g. PERK kinases, MLO-like receptors), carbohydrate active enzymes (e.g. XTH18), transcription factors (e.g. members of ZIM, WRKY, NAC), and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1). We verified the NaClinducible expression of selected transcription factors and other genes by qRT-PCR. Conclusion: Micorarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent transcriptional responses to stress, will facilitate mapping of regulatory networks and extend our ability to improve salt tolerance in plants.

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  • Length CDNA Microarray

    • High-throughput, high-sensitivity analysis of gene expression in Arabidopsis

      High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes.

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  • Oryza-Sativa-L

    • Gene expression profiles during the initial phase of salt stress in rice

      Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defenserelated functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.

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  • Pathogenesis-related proteins

    • Transcriptional profiling of pea ABR17 mediated changes in gene expression in Arabidopsis thaliana

      Background: Pathogenesis-related proteins belonging to group 10 (PR10) are elevated in response to biotic and abiotic stresses in plants. Previously, we have shown a drastic salinityinduced increase in the levels of ABR17, a member of the PR10 family, in pea. Furthermore, we have also demonstrated that the constitutive expression of pea ABR17 cDNA in Arabidopsis thaliana and Brassica napus enhances their germination and early seedling growth under stress. Although it has been reported that several members of the PR10 family including ABR17 possess RNase activity, the exact mechanism by which the aforementioned characteristics are conferred by ABR17 is unknown at this time. We hypothesized that a study of differences in transcriptome between wild type (WT) and ABR17 transgenic A. thaliana may shed light on this process. Results: The molecular changes brought about by the expression of pea ABR17 cDNA in A. thaliana in the presence or absence of salt stress were investigated using microarrays consisting of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes. Statistical analysis identified number of genes which were over represented among up- or down-regulated transcripts in the transgenic line. Our results highlight the important roles of many abscisic acid (ABA) and cytokinin (CK) responsive genes in ABR17 transgenic lines. Although the transcriptional changes followed a general salt response theme in both WT and transgenic seedlings under salt stress, many genes exhibited differential expression patterns when the transgenic and WT lines were compared. These genes include plant defensins, heat shock proteins, other defense related genes, and several transcriptional factors. Our microarray results for selected genes were validated using quantitative real-time PCR. Conclusion: Transcriptional analysis in ABR17 transgenic Arabidopsis plants, both under normal and saline conditions, revealed significant changes in abundance of transcripts for many stress responsive genes, as well as those related to plant growth and development. Our results also suggest that ABR17 may mediate stress tolerance through the modulation of many ABA- and CKresponsive genes and may further our understanding of the role of ABR17 in mediating plant stress responses.

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  • RAPD analysis

    • Molecular Diversity of Ascochyta rabiei Isolates from Chickpea in Alberta, Canada

      During the summer of 2003 and 2004, a total of 58 isolates of Ascochyta rabiei were collected from chickpea plants grown in southern Alberta, Canada. RAPD analysis of genomic DNA extracted from these isolates was conducted using six short sequence primers (OPA-03, OPA-13, OPB-07, OPC-01, OPC-20 and OPJ-15) and analyzed to establish the genetic relationship and distance between isolates. A total of 75 bands were polymorphic. The 58 isolates were found to belong to five genotypes, indicating that the A. rabiei population of southern Alberta is genetically diverse. No relationship was found between the genotype groupings obtained through RAPD analysis and previously determined pathotype classifications of the same isolates.

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  • Systemic aquired-resistance

    • Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments

      Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses. Results: In this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only. The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns. Conclusion: We identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

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