Caroline Levasseur

Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis

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.

EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar

• The eucalyptus R2R3 transcription factor, EgMYB1 contains an active repressor motif in the regulatory domain of the predicted protein. It is preferentially expressed in differentiating xylem and is capable of repressing the transcription of two key lignin genes in vivo. • In order to investigate in planta the role of this putative transcriptional repressor of the lignin biosynthetic pathway, we overexpressed the EgMYB1 gene in Arabidopsis and poplar. • Expression of EgMYB1 produced similar phenotypes in both species, with stronger effects in transgenic Arabidopsis plants than in poplar. Vascular development was altered in overexpressors showing fewer lignified fibres (in phloem and interfascicular zones in poplar and Arabidopsis, respectively) and reduced secondary wall thickening. Klason lignin content was moderately but significantly reduced in both species. Decreased transcript accumulation was observed for genes involved in the biosynthesis of lignins, cellulose and xylan, the three main polymers of secondary cell walls. Transcriptomic profiles of transgenic poplars were reminiscent of those reported when lignin biosynthetic genes are disrupted. • Together, these results strongly suggest that EgMYB1 is a repressor of secondary wall formation and provide new opportunities to dissect the transcriptional regulation of secondary wall biosynthesis.

Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses

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.

Expression profiling and functional analysis of Populus WRKY23 reveals a regulatory role in defense

WRKY transcription factors are key regulators that activate and fine-tune stress responses, including defense responses against pathogens. We isolated a poplar (Populus tremulaxPopulus alba) cDNA sequence, PtWRKY23, that encodes the ortholog of Arabidopsis WRKY23 and present the functional analysis of WRKY23, with emphasis on its potential role in resistance to rust infection. To investigate the function of PtWRKY23, we examined PtWRKY23 expression after stress treatments by qRT-PCR and generated PtWRKY23-misexpressing plants. Transgenic plants were assessed for resistance to Melampsora rust and were analyzed using the poplar Affymetrix GeneChip and histological techniques to study the consequences of PtWRKY23 misexpression. PtWRKY23 is rapidly induced by Melampsora infection and elicitor treatments and poplars overexpressing and underexpressing PtWRKY23 were both more susceptible to Melampsora infection than wild type. Transcriptome analysis of PtWRKY23 overexpressors revealed a significant overlap with the Melampsora-infection response. Transcriptome analysis also suggests that PtWRKY23 affects redox homeostasis and cell wall-related metabolism, which was confirmed by analyses that showed that PtWRKY23-misexpressing plants have altered peroxidase activity, apparent H(2)O(2) accumulation and lignin deposition. Our results show that PtWRKY23 affects resistance to Melampsora infection and that this may be caused by deregulation of genes that disrupt redox homeostasis and cell wall metabolism.

Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees.

BackgroundClass 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.ResultsFull-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.ConclusionsThis 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.

Sequence analysis and functional characterization of the promoter of the Picea glauca Cinnamyl Alcohol Dehydrogenase gene in transgenic white spruce plants.

The enzyme CinnamylAlcoholDehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5′ flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Piceaglauca (Moench) Voss) and loblolly pine (Pinustaeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P.glaucaCAD promoter used P.glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the β-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood.

Testing of a heterologous, wound- and insect-inducible promoter for functional genomics studies in conifer defense

Large-scale sequencing of conifer cDNAs and targeted molecular cloning have identified many putative conifer defense genes. Expression of many of these genes is induced in response to biotic stress and some may be expressed only in a few specialized tissues or cells. Proven functional genomics approaches to test these genes involve expression of proteins in Escherichia coli or yeast for biochemical characterization or constitutive over-expression in transformed plants. Plant transformation to test functions of insect-, wound- or pathogen-induced conifer defense genes would benefit from the use of an inducible expression system. We describe here the development of a heterologous, wound- and insect-inducible gene expression system for conifers using the potato proteinase inhibitor II (pinII)-promoter. Using pinII::GUS and pinII::(E)-α-bisabolene synthase expression constructs we demonstrate localized induced gene expression in white spruce seedlings (Picea glauca). Testing of these constructs in Arabidopsis thaliana and tobacco illustrates the additional potential of the pinII-promoter to be used in tests of gene function that involve cell-specific and systemic induction.

Evidence of Somaclonal Variation in Somatic Embryo-Derived Plantlets of White Spruce (Picea Glauca (Moench) Voss

Unlike angiosperms (especially crop plant species), conifers are considered to be genetically stable following somatic embryogenesis. However, we have been able to identify four different variegata phenotypes among 2270 somatic embryo-derived white spruces. The four types of variegated plants differ from each other with respect to the extent and distribution of their chlorophyll-deficient needles. Microscopy shows that certain leaves of a selected variant are formed of a chimeral mixture of green and white cells. Cells in completely white needles of this variegated plant are characterized by large nuclei with predominant euchromatin, absence of large cytoplasmic vacuoles, and vacuolized plastids with aberrant morphologies. Various observations suggest that the recovered variegata phenotypes reflect some kind of genetic instability of either chloroplastic or nuclear genomes. Molecular approaches, including the use of RAPD markers, are currently employed to find out whether DNA rearrangements are involved in conferring these variegata phenotypes.

Development of a Traceability System Based on a SNP Array for Large-Scale Production of High-Value White Spruce (Picea glauca)

Biological material is at the forefront of research programs, as well as application fields such as breeding, aquaculture, and reforestation. While sophisticated techniques are used to produce this material, all too often, there is no strict monitoring during the “production” process to ensure that the specific varieties are the expected ones. Confidence rather than evidence is often applied when the time comes to start a new experiment or to deploy selected varieties in the field. During the last decade, genomics research has led to the development of important resources, which have created opportunities for easily developing tools to assess the conformity of the material along the production chains. In this study, we present a simple methodology that enables the development of a traceability system which, is in fact a by-product of previous genomic projects. The plant production system in white spruce (Picea glauca) is used to illustrate our purpose. In Quebec, one of the favored strategies to produce elite varieties is to use somatic embryogenesis (SE). In order to detect human errors both upstream and downstream of the white spruce production process, this project had two main objectives: (i) to develop methods that make it possible to trace the origin of plants produced, and (ii) to generate a unique genetic fingerprint that could be used to differentiate each embryogenic cell line and ensure its genetic monitoring. Such a system had to rely on a minimum number of low-cost DNA markers and be easy to use by non-specialists. An efficient marker selection process was operationalized by testing different classification methods on simulated datasets. These datasets were generated using in-house bioinformatics tools that simulated crosses involved in the breeding program for which genotypes from hundreds of SNP markers were already available. The rate of misidentification was estimated and various sources of mishandling or contamination were identified. The method can easily be applied to other production systems for which genomic resources are already available.