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. 2017 Oct;216(2):482-494.
doi: 10.1111/nph.14458. Epub 2017 Feb 10.

NorWood: A Gene Expression Resource for Evo-Devo Studies of Conifer Wood Development

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Free PMC article

NorWood: A Gene Expression Resource for Evo-Devo Studies of Conifer Wood Development

Soile Jokipii-Lukkari et al. New Phytol. .
Free PMC article

Abstract

The secondary xylem of conifers is composed mainly of tracheids that differ anatomically and chemically from angiosperm xylem cells. There is currently no high-spatial-resolution data available profiling gene expression during wood formation for any coniferous species, which limits insight into tracheid development. RNA-sequencing data from replicated, high-spatial-resolution section series throughout the cambial and woody tissues of Picea abies were used to generate the NorWood.conGenIE.org web resource, which facilitates exploration of the associated gene expression profiles and co-expression networks. Integration within PlantGenIE.org enabled a comparative regulomics analysis, revealing divergent co-expression networks between P. abies and the two angiosperm species Arabidopsis thaliana and Populus tremula for the secondary cell wall (SCW) master regulator NAC Class IIB transcription factors. The SCW cellulose synthase genes (CesAs) were located in the neighbourhoods of the NAC factors in A. thaliana and P. tremula, but not in P. abies. The NorWood co-expression network enabled identification of potential SCW CesA regulators in P. abies. The NorWood web resource represents a powerful community tool for generating evo-devo insights into the divergence of wood formation between angiosperms and gymnosperms and for advancing understanding of the regulation of wood development in P. abies.

Keywords: Norway spruce (Picea abies); RNA-sequencing; co-expression network; comparative genomics; cryosection; evo-devo; growth ring; secondary cell wall.

Figures

Figure 1
Figure 1
Clustering of Picea abies gene expression profiles matching developmental transitions. (a) A representative transverse section stained with nitroblue tetrazolium (NBT) from the stem of Tree 1. A high‐spatial‐resolution gene expression atlas was created by collecting series of tangential cryosections from the stem. The sections were pooled into 14–18 pools in each tree in such a way that representative samples were obtained for each stage of xylem development including cambium (C), xylem expansion (EX), secondary cell wall formation (SCW), programmed cell death (PCD), mature xylem (MX) and the previous year′s latewood formation (LW). Bar, 200 μm. (b) Hierarchical clustering of gene expression data in cryosection series of three trees. Seven gene expression clusters are indicated (a–f, z). Cluster‐z contained genes with no defined profile across the cutting series and was not further analysed. Samples appear in the order of sampling within each tree (T1–T3) and three sample clusters are indicated (blue, red and brown). Expression values are scaled per gene so that expression values above the gene average are represented by red, and below average by blue. (c) Expression profiles of genes within six of the gene clusters. The average expression of each gene cluster is shown in white with all expression profiles from Tree 1 plotted in grey in the background. (d) Genes were assigned to the sample(s) within Tree 1 in which they were expressed within 4% of the maximum expression for that gene, after which gene ontology (GO) enrichment was calculated for each sample. Selected GO categories are represented. All significant categories are available in Supporting Information Table S5.
Figure 2
Figure 2
Using the NorWood resource to identify putative regulators of secondary cell wall cellulose synthase genes. (a) Arabidopsis thaliana cellulose synthase A7 (CesA7; AT5G17420) was used to find and retrieve the cellulose synthase gene family at AtGenIE.org. A phylogenetic tree of the CesA gene family for A. thaliana, Picea abies (Norway spruce) and Populus trichocarpa (poplar) produced using the PlantGenIE phylogenetic tree galaxy workflow is shown. (b) Conservation of co‐expression between the expressed CesA gene family members in NorWood and their P. trichocarpa orthologues was examined using the complex tool. (c) The network neighbourhood of CesA genes with conserved co‐expression was retrieved using the NorWood dataset within the exNet tool at ConGenIE using a context‐likelihood of relatedness (CLR) threshold of 5. This neighbourhood contained four genes comprising MA_183130g0010, MA_140410g0010 and MA_10429177g0010, which are orthologous to the A. thaliana secondary cell wall CesA genes, and one secondary cell wall CesA‐like gene, MA_10429339g0020. Shown in this panel on the left is the network, in the centre are line plots from the explot tool with one coloured line representing each of the four neighbourhood genes and the network neighbourhood of these four genes on the right. In the line plots, T1–T3 represent the three replicate trees and dashed vertical lines represent cambium/expanding xylem, secondary cell wall/programmed cell death and mature xylem/late wood zones. (d) The subset of network neighbours annotated as transcription factors (TFs) were selected. The network neighbourhood of this TF subset was then selected at a CLR threshold of 5, subset to genes annotated as TFs, the expression of which were visualised as a heatmap within the NorWood resource. Shown on the left are the starting nine TFs identified in (c), in the centre is the network of TFs where the original nine are shown in green and where neighbours connected by positive edges (i.e. a positive Pearson correlation, indicated as grey edges) are shown in pink and those connected by negative edges (red edges) are shown in blue. In the heatmap expression values are scaled per gene so that expression values above the gene average are represented by red, and below average by blue. Samples appear in the order of sampling within each tree (T1–T3).
Figure 3
Figure 3
The network neighbourhood of the Class IIB NAC family indicates divergence in the regulation of cellulose synthase genes. (a) A phylogenetic tree of the Class IIB NAC family. Two of the four Picea abies genes were expressed in the NorWood dataset and are highlighted in blue and red. (b) The AtGenExpress Development (Schmid et al., 2005) network neighbourhood of the Arabidopsis thaliana NAC IIB subfamily was identified using the AtGenIE exnet tool at a context‐likelihood of relatedness (CLR) threshold of 5. Secondary cell wall cellulose synthase genes (CesA4, 7 and 8) are indicated. (c) The Populus tremula exAtlas (Sundell et al., 2015) network neighbourhood of the Populus trichocarpa NAC IIB family was identified using the PopGenIE exnet tool at a CLR threshold of 5. (d) The Pabies exAtlas (Nystedt et al., 2013) network neighbourhood of the NAC IIB subfamily was identified using the ConGenIE exnet tool at a CLR threshold of 3.5. A lower CLR threshold was used to ensure a network of comparable size. (e) The NorWood network neighbourhood of the P. abies NAC IIB subfamily was identified using the ConGenIE exnet tool at a CLR threshold of 3.5. The NST and VND orthologues reside in different subnetworks connected with negatively correlated expression profiles (red links). No CesA genes were present in the neighbourhood of NST or of VND. (f) Gene expression profiles of Pabies VND and NST and heatmap representations of their respective positive network neighbourhood within the NorWood co‐expression network (CLR threshold 5). For the VST and NST homologue, the expression profile is shown for the three replicate trees (T1–T3) where dashed vertical lines represent cambium/expanding xylem, secondary cell wall/programmed cell death and mature xylem/late wood zones. A heatmap of the corresponding network neighbourhood is shown below the expression profile plots with expression values scaled per gene so that expression values above the gene average are represented by red, and below average by blue. Samples appear in the order of sampling within each tree (T1–T3).

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