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1.  Opposite action of R2R3-MYBs from different subgroups on key genes of the shikimate and monolignol pathways in spruce 
Journal of Experimental Botany  2013;65(2):495-508.
Redundancy and competition between R2R3-MYB activators and repressors on common target genes has been proposed as a fine-tuning mechanism for the regulation of plant secondary metabolism. This hypothesis was tested in white spruce [Picea glauca (Moench) Voss] by investigating the effects of R2R3-MYBs from different subgroups on common targets from distinct metabolic pathways. Comparative analysis of transcript profiling data in spruces overexpressing R2R3-MYBs from loblolly pine (Pinus taeda L.), PtMYB1, PtMYB8, and PtMYB14, defined a set of common genes that display opposite regulation effects. The relationship between the closest MYB homologues and 33 putative target genes was explored by quantitative PCR expression profiling in wild-type P. glauca plants during the diurnal cycle. Significant Spearman’s correlation estimates were consistent with the proposed opposite effect of different R2R3-MYBs on several putative target genes in a time-related and tissue-preferential manner. Expression of sequences coding for 4CL, DHS2, COMT1, SHM4, and a lipase thio/esterase positively correlated with that of PgMYB1 and PgMYB8, but negatively with that of PgMYB14 and PgMYB15. Complementary electrophoretic mobility shift assay (EMSA) and transactivation assay provided experimental evidence that these different R2R3-MYBs are able to bind similar AC cis-elements in the promoter region of Pg4CL and PgDHS2 genes but have opposite effects on their expression. Competitive binding EMSA experiments showed that PgMYB8 competes more strongly than PgMYB15 for the AC-I MYB binding site in the Pg4CL promoter. Together, the results bring a new perspective to the action of R2R3-MYB proteins in the regulation of distinct but interconnecting metabolism pathways.
doi:10.1093/jxb/ert398
PMCID: PMC3904711  PMID: 24336492
Conifers; phenylpropanoid pathway; protein–DNA binding; R2R3-MYB evolution; transcriptional network.
2.  Molecular and microscopic analysis of the gut contents of abundant rove beetle species (Coleoptera, Staphylinidae) in the boreal balsam fir forest of Quebec, Canada 
ZooKeys  2013;1-24.
Experimental research on beetle responses to removal of logging residues following clearcut harvesting in the boreal balsam fir forest of Quebec revealed several abundant rove beetle (Staphylinidae) species potentially important for long-term monitoring. To understand the trophic affiliations of these species in forest ecosystems, it was necessary to analyze their gut contents. We used microscopic and molecular (DNA) methods to identify the gut contents of the following rove beetles: Atheta capsularis Klimaszewski, Atheta klagesi Bernhauer, Oxypoda grandipennis (Casey), Bryophacis smetanai Campbell, Ischnosoma longicorne (Mäklin), Mycetoporus montanus Luze, Tachinus frigidus Erichson, Tachinus fumipennis (Say), Tachinus quebecensis Robert, and Pseudopsis subulata Herman. We found no apparent arthropod fragments within the guts; however, a number of fungi were identified by DNA sequences, including filamentous fungi and budding yeasts [Ascomycota: Candida derodonti Suh & Blackwell (accession number FJ623605), Candida mesenterica (Geiger) Diddens & Lodder (accession number FM178362), Candida railenensis Ramirez and Gonzáles (accession number JX455763), Candida sophie-reginae Ramirez & González (accession number HQ652073), Candida sp. (accession number AY498864), Pichia delftensis Beech (accession number AY923246), Pichia membranifaciens Hansen (accession number JQ26345), Pichia misumaiensis Y. Sasaki and Tak. Yoshida ex Kurtzman 2000 (accession number U73581), Pichia sp. (accession number AM261630), Cladosporium sp. (accession number KF367501), Acremoniumpsammosporum W. Gams (accession number GU566287), Alternaria sp. (accession number GU584946), Aspergillus versicolor Bubak (accession number AJ937750), and Aspergillusamstelodami (L. Mangin) Thom and Church (accession number HQ728257)]. In addition, two species of bacteria [Bradyrhizobium japonicum (Kirchner) Jordan (accession number BA000040) and Serratia marcescens Bizio accession number CP003942] were found in the guts. These results not only provide evidence of the consumer-resource relations of these beetles but also clarify the relationship between rove beetles, woody debris and fungi. Predominance of yeast-feeding by abundant rove beetles suggests that it may play an important role in their dietary requirements.
doi:10.3897/zookeys.353.5991
PMCID: PMC3837482  PMID: 24294095
Rove beetles; Staphylinidae; Coleoptera; diet; fungivory; mycophagy; gut analysis; trophic relationship; saproxylic; boreal forest; Canada; Ascomycota; Basidiomycota; bacteria
3.  The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms* 
Journal of Experimental Botany  2011;63(2):785-795.
A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.
doi:10.1093/jxb/err303
PMCID: PMC3254680  PMID: 22048038
Arabidopsis; Ceratocystis; defensin; jasmonic acid; Picea glauca; promoter; wounding
7.  Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees 
BMC Plant Biology  2010;10:273.
Background
Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups.
Results
Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with the functions that have been assigned to close homologs in herbaceous plants.
Conclusions
This study provides an overview of HD-zip III genes related to woody plant development and identifies sequences putatively involved in secondary vascular growth in angiosperms and in gymnosperms. These gene sequences are candidate regulators of wood formation and could be a source of molecular markers for tree breeding related to wood properties.
doi:10.1186/1471-2229-10-273
PMCID: PMC3017839  PMID: 21143995
8.  Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar 
BMC Genomics  2010;11:674.
Background
Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development.
Results
We report that poplar (Populus spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast.
Conclusion
These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.
doi:10.1186/1471-2164-11-674
PMCID: PMC3091788  PMID: 21114852
9.  Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses 
Journal of Experimental Botany  2010;61(14):3847-3864.
Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-represention of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitiative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution.
doi:10.1093/jxb/erq196
PMCID: PMC2935864  PMID: 20732878
Gene family expansion; gymnosperms; isoprenoid metabolism; MYB transcription factors; microarray RNA profiling; Picea glauca; plant evolution; stress response; terpenes; tissue-specific expression
10.  Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community ▿ †  
Applied and Environmental Microbiology  2009;75(23):7527-7536.
The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.
doi:10.1128/AEM.01120-09
PMCID: PMC2786396  PMID: 19801471
11.  Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis 
Journal of Experimental Botany  2008;59(14):3925-3939.
The involvement of two R2R3-MYB genes from Pinus taeda L., PtMYB1 and PtMYB8, in phenylpropanoid metabolism and secondary cell wall biogenesis was investigated in planta. These pine MYBs were constitutively overexpressed (OE) in Picea glauca (Moench) Voss, used as a heterologous conifer expression system. Morphological, histological, chemical (lignin and soluble phenols), and transcriptional analyses, i.e. microarray and reverse transcription quantitative PCR (RT-qPCR) were used for extensive phenotyping of MYB-overexpressing spruce plantlets. Upon germination of somatic embryos, root growth was reduced in both transgenics. Enhanced lignin deposition was also a common feature but ectopic secondary cell wall deposition was more strongly associated with PtMYB8-OE. Microarray and RT-qPCR data showed that overexpression of each MYB led to an overlapping up-regulation of many genes encoding phenylpropanoid enzymes involved in lignin monomer synthesis, while misregulation of several cell wall-related genes and other MYB transcription factors was specifically associated with PtMYB8-OE. Together, the results suggest that MYB1 and MYB8 may be part of a conserved transcriptional network involved in secondary cell wall deposition in conifers.
doi:10.1093/jxb/ern234
PMCID: PMC2576632  PMID: 18805909
12.  MAP-ping genomic organization and organ-specific expression profiles of poplar MAP kinases and MAP kinase kinases 
BMC Genomics  2006;7:223.
Background
As in other eukaryotes, plant mitogen-activated protein kinase (MAPK) cascades are composed of three classes of hierarchically organized protein kinases, namely MAPKKKs, MAPKKs, and MAPKs. These modules rapidly amplify and transduce extracellular signals into various appropriate intracellular responses. While extensive work has been conducted on the post-translational regulation of specific MAPKKs and MAPKs in various plant species, there has been no systematic investigation of the genomic organization and transcriptional regulation of these genes.
Results
Ten putative poplar MAPKK genes (PtMKKs) and 21 putative poplar MAPK genes (PtMPKs) have been identified and located within the poplar (Populus trichocarpa) genome. Analysis of exon-intron junctions and of intron phase inside the predicted coding region of each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Expression profiles of all members of these two gene families were also analyzed in 17 different poplar organs, using gene-specific primers directed at the 3'-untranslated region of each candidate gene and real-time quantitative PCR. Most PtMKKs and PtMPKs were differentially expressed across this developmental series.
Conclusion
This analysis provides a complete survey of MAPKK and MAPK gene expression profiles in poplar, a large woody perennial plant, and thus complements the extensive expression profiling data available for the herbaceous annual Arabidopsis thaliana. The poplar genome is marked by extensive segmental and chromosomal duplications, and within both kinase families, some recently duplicated paralogous gene pairs often display markedly different patterns of expression, consistent with the rapid evolution of specialized protein functions in this highly adaptive species.
doi:10.1186/1471-2164-7-223
PMCID: PMC1574314  PMID: 16945144
13.  In Vivo Monitoring of Obligate Biotrophic Pathogen Growth by Kinetic PCR 
The plant kingdom is constantly challenged by a battery of evolving pathogens. New species or races of pathogens are discovered on crops that were initially bred for disease resistance, and globalization is facilitating the movement of exotic pests. Among these pests, obligate biotrophic parasites make up some of the most damaging groups and have been particularly challenging to study. Here we demonstrate the utility of kinetic PCR (kPCR) (real-time PCR, quantitative PCR) to assess the growth of poplar rust, caused by Melampsora species, by quantification of pathogen DNA. kPCR allowed the construction of reliable growth curves from inoculation through the final stages of uredinial maturation, as well as pathogen monitoring before symptoms become visible. Growth parameters, such as latency period, generation time in logarithmic growth, and the increase in DNA mass at saturation, were compared in compatible, incompatible, and nonhost interactions. Pathogen growth was monitored in different applications dealing with plant pathology, such as host and pathogen diversity and transgenic crop improvement. Finally, the capacity of kPCR to differentiate pathogens in the same sample has broad molecular ecology applications for dynamically monitoring the growth of fungi in their environments or in mixed populations or to measure the efficacy of pest control strategies.
doi:10.1128/AEM.71.3.1546-1552.2005
PMCID: PMC1065157  PMID: 15746359
14.  Genetically engineered trees for plantation forests: key considerations for environmental risk assessment 
Plant Biotechnology Journal  2013;11(7):785-798.
Forests are vital to the world's ecological, social, cultural and economic well-being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial-scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter-lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.
doi:10.1111/pbi.12100
PMCID: PMC3823068  PMID: 23915092
genetic engineering; forests; environment; risk assessment

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