A transcriptome atlas of zygotic and somatic embryogenesis in Norway spruce.

Somatic embryogenesis (SE) is a powerful model system for studying embryo development and an important method for scaling up availability of elite and climate-adapted genetic material of Norway spruce (Picea abies L. Karst). However, there are several steps during the development of the somatic embryo (Sem) that are suboptimal compared to zygotic embryo (Zem) development. These differences are poorly understood and result in substantial yield losses during plant production, which limits cost-effective large-scale production of SE plants. This study presents a comprehensive data resource profiling gene expression during zygotic and somatic embryo development to support studies aiming to advance understanding of gene regulatory programmes controlling embryo development. Transcriptome expression patterns were analysed during zygotic embryogenesis (ZE) in Norway spruce, including separated samples of the female gametophytes and Zem, and at multiple stages during SE. Expression data from eight developmental stages of SE, starting with pro-embryogenic masses (PEMs) up until germination, revealed extensive modulation of the transcriptome between the early and mid-stage maturing embryos and at the transition of desiccated embryos to germination. Comparative analysis of gene expression changes during ZE and SE identified differences in the pattern of gene expression changes and functional enrichment of these provided insight into the associated biological processes. Orthologs of transcription factors known to regulate embryo development in angiosperms were differentially regulated during Zem and Sem development and in the different zygotic embryo tissues, providing clues to the differences in development observed between Zem and Sem. This resource represents the most comprehensive dataset available for exploring embryo development in conifers.

A resource of identified and annotated lincRNAs expressed during somatic embryogenesis development in Norway spruce.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of many biological processes, although their regulatory roles remain poorly characterized in woody plants, especially in gymnosperms. A major challenge of working with lncRNAs is to assign functional annotations, since they have a low coding potential and low cross-species conservation. We utilised an existing RNA-Sequencing resource and performed short RNA sequencing of somatic embryogenesis developmental stages in Norway spruce (Picea abies L. Karst). We implemented a pipeline to identify lncRNAs located within the intergenic space (lincRNAs) and generated a co-expression network including protein coding, lincRNA and miRNA genes. To assign putative functional annotation, we employed a guilt-by-association approach using the co-expression network and integrated these results with annotation assigned using semantic similarity and co-expression. Moreover, we evaluated the relationship between lincRNAs and miRNAs, and identified which lincRNAs are conserved in other species. We identified lincRNAs with clear evidence of differential expression during somatic embryogenesis and used network connectivity to identify those with the greatest regulatory potential. This work provides the most comprehensive view of lincRNAs in Norway spruce and is the first study to perform global identification of lincRNAs during somatic embryogenesis in conifers. The data have been integrated into the expression visualisation tools at the PlantGenIE.org web resource to enable easy access to the community. This will facilitate the use of the data to address novel questions about the role of lincRNAs in the regulation of embryogenesis and facilitate future comparative genomics studies.

An Improved Chromosome-scale Genome Assembly and Population Genetics resource for Populus tremula.

Aspen (Populus tremula L.) is a keystone species and a model system for forest tree genomics. We present an updated resource comprising a chromosome-scale assembly, population genetics and genomics data. Using the resource, we explore the genetic basis of natural variation in leaf size and shape, traits with complex genetic architecture. We generated the genome assembly using long-read sequencing, optical and high-density genetic maps. We conducted whole-genome resequencing of the Umeå Aspen (UmAsp) collection. Using the assembly and re-sequencing data from the UmAsp, Swedish Aspen (SwAsp) and Scottish Aspen (ScotAsp) collections we performed genome-wide association analyses (GWAS) using Single Nucleotide Polymorphisms (SNPs) for 26 leaf physiognomy phenotypes. We conducted Assay of Transposase Accessible Chromatin sequencing (ATAC-Seq), identified genomic regions of accessible chromatin, and subset SNPs to these regions, improving the GWAS detection rate. We identified candidate long non-coding RNAs in leaf samples, quantified their expression in an updated co-expression network, and used this to explore the functions of candidate genes identified from the GWAS. A GWAS found SNP associations for seven traits. The associated SNPs were in or near genes annotated with developmental functions, which represent candidates for further study. Of particular interest was a ~177-kbp region harbouring associations with several leaf phenotypes in ScotAsp. We have incorporated the assembly, population genetics, genomics, and GWAS data into the PlantGenIE.org web resource, including updating existing genomics data to the new genome version, to enable easy exploration and visualisation. We provide all raw and processed data to facilitate reuse in future studies.

Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

Metabolic control of arginine and ornithine levels paces the progression of leaf senescence.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

SeedTransNet: a directional translational network revealing regulatory patterns during seed maturation and germination.

Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

An atlas of the Norway spruce needle seasonal transcriptome.

Boreal conifers possess a tremendous ability to survive and remain evergreen during harsh winter conditions and resume growth during summer. This is enabled by coordinated regulation of major cellular functions at the level of gene expression, metabolism, and physiology. Here we present a comprehensive characterization of the annual changes in the global transcriptome of Norway spruce (Picea abies) needles as a resource to understand needle development and acclimation processes throughout the year. In young, growing needles (May 15 until June 30), cell walls, organelles, etc., were formed, and this developmental program heavily influenced the transcriptome, explained by over-represented Gene Ontology (GO) categories. Later changes in gene expression were smaller but four phases were recognized: summer (July-August), autumn (September-October), winter (November-February), and spring (March-April), where over-represented GO categories demonstrated how the needles acclimated to the various seasons. Changes in the seasonal global transcriptome profile were accompanied by differential expression of members of the major transcription factor families. We present a tentative model of how cellular activities are regulated over the year in needles of Norway spruce, which demonstrates the value of mining this dataset, accessible in ConGenIE together with advanced visualization tools.

ProkSeq for complete analysis of RNA-Seq data from prokaryotes.

SUMMARY: Since its introduction, RNA-Seq technology has been used extensively in studies of pathogenic bacteria to identify and quantify differences in gene expression across multiple samples from bacteria exposed to different conditions. With some exceptions, tools for studying gene expression, determination of differential gene expression, downstream pathway analysis and normalization of data collected in extreme biological conditions is still lacking. Here, we describe ProkSeq, a user-friendly, fully automated RNA-Seq data analysis pipeline designed for prokaryotes. ProkSeq provides a wide variety of options for analysing differential expression, normalizing expression data and visualizing data and results. AVAILABILITY AND IMPLEMENTATION: ProkSeq is implemented in Python and is published under the MIT source license. The pipeline is available as a Docker container https://hub.docker.com/repository/docker/snandids/prokseq-v2.0, or can be used through Anaconda: https://anaconda.org/snandiDS/prokseq. The code is available on Github: https://github.com/snandiDS/prokseq and a detailed user documentation, including a manual and tutorial can be found at https://prokseqV20.readthedocs.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula × Populus tremuloides.

The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin-mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in Laccaria bicolor for ECM formation with Populus tremula × Populus tremuloides. We combine transcriptomics of cell-wall-related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic L. bicolor to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation. Immunolocalisation identified remodelling of pectin towards de-esterified HG during ECM formation, which was accompanied by increased LbPME1 expression and PME activity. Overexpression or RNAi of the ECM-induced LbPME1 in transgenic L. bicolor lines led to reduced ECM formation. Hartig Nets formed with LbPME1 RNAi lines were shallower, whereas those formed with LbPME1 overexpressors were deeper. This suggests that LbPME1 plays a role in ECM formation potentially through HG de-esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation.

Cone-setting in spruce is regulated by conserved elements of the age-dependent flowering pathway.

Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV.

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes' ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

qtlXplorer: an online systems genetics browser in the Eucalyptus Genome Integrative Explorer (EucGenIE).

BACKGROUND: Affordable high-throughput DNA and RNA sequencing technologies are allowing genomic analysis of plant and animal populations and as a result empowering new systems genetics approaches to study complex traits. The availability of intuitive tools to browse and analyze the resulting large-scale genetic and genomic datasets remain a significant challenge. Furthermore, these integrative genomics approaches require innovative methods to dissect the flow and interconnectedness of biological information underlying complex trait variation. The Plant Genome Integrative Explorer (PlantGenIE.org) is a multi-species database and domain that houses online tools for model and woody plant species including Eucalyptus. Since the Eucalyptus Genome Integrative Explorer (EucGenIE) is integrated within PlantGenIE, it shares genome and expression analysis tools previously implemented within the various subdomains (ConGenIE, PopGenIE and AtGenIE). Despite the success in setting up integrative genomics databases, online tools for systems genetics modelling and high-resolution dissection of complex trait variation in plant populations have been lacking. RESULTS: We have developed qtlXplorer ( https://eucgenie.org/QTLXplorer ) for visualizing and exploring systems genetics data from genome-wide association studies including quantitative trait loci (QTLs) and expression-based QTL (eQTL) associations. This module allows users to, for example, find co-located QTLs and eQTLs using an interactive version of Circos, or explore underlying genes using JBrowse. It provides users with a means to build systems genetics models and generate hypotheses from large-scale population genomics data. We also substantially upgraded the EucGenIE resource and show how it enables users to combine genomics and systems genetics approaches to discover candidate genes involved in biotic stress responses and wood formation by focusing on two multigene families, laccases and peroxidases. CONCLUSIONS: qtlXplorer adds a new dimension, population genomics, to the EucGenIE and PlantGenIE environment. The resource will be of interest to researchers and molecular breeders working in Eucalyptus and other woody plant species. It provides an example of how systems genetics data can be integrated with functional genetics data to provide biological insight and formulate hypotheses. Importantly, integration within PlantGenIE enables novel comparative genomics analyses to be performed from population-scale data.

Functional and evolutionary genomic inferences in Populus through genome and population sequencing of American and European aspen.

The Populus genus is one of the major plant model systems, but genomic resources have thus far primarily been available for poplar species, and primarily Populus trichocarpa (Torr. & Gray), which was the first tree with a whole-genome assembly. To further advance evolutionary and functional genomic analyses in Populus, we produced genome assemblies and population genetics resources of two aspen species, Populus tremula L. and Populus tremuloides Michx. The two aspen species have distributions spanning the Northern Hemisphere, where they are keystone species supporting a wide variety of dependent communities and produce a diverse array of secondary metabolites. Our analyses show that the two aspens share a similar genome structure and a highly conserved gene content with P. trichocarpa but display substantially higher levels of heterozygosity. Based on population resequencing data, we observed widespread positive and negative selection acting on both coding and noncoding regions. Furthermore, patterns of genetic diversity and molecular evolution in aspen are influenced by a number of features, such as expression level, coexpression network connectivity, and regulatory variation. To maximize the community utility of these resources, we have integrated all presented data within the PopGenIE web resource (PopGenIE.org).

Poplar carbohydrate-active enzymes: whole-genome annotation and functional analyses based on RNA expression data.

Carbohydrate-active enzymes (CAZymes) catalyze the formation and modification of glycoproteins, glycolipids, starch, secondary metabolites and cell wall biopolymers. They are key enzymes for the biosynthesis of food and renewable biomass. Woody biomass is particularly important for long-term carbon storage and as an abundant renewable natural resource for many industrial applications. This study presents a re-annotation of CAZyme genes in the current Populus trichocarpa genome assembly and in silico functional characterization, based on high-resolution RNA-Seq data sets. Altogether, 1914 CAZyme and expansin genes were annotated in 101 families. About 1797 of these genes were found expressed in at least one Populus organ. We identified genes involved in the biosynthesis of different cell wall polymers and their paralogs. Whereas similar families exist in poplar and Arabidopsis thaliana (with the exception of CBM13 found only in poplar), a few families had significantly different copy numbers between the two species. To identify the transcriptional coordination and functional relatedness within the CAZymes and other proteins, we performed co-expression network analysis of CAZymes in wood-forming tissues using the AspWood database (http://aspwood.popgenie.org/aspwood-v3.0/) for Populus tremula. This provided an overview of the transcriptional changes in CAZymes during the transition from primary to secondary wall formation, and the clustering of transcripts into potential regulons. Candidate enzymes involved in the biosynthesis of polysaccharides were identified along with many tissue-specific uncharacterized genes and transcription factors. These collections offer a rich source of targets for the modification of secondary cell wall biosynthesis and other developmental processes in woody plants.

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce.

A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

PECTIN ACETYLESTERASE9 Affects the Transcriptome and Metabolome and Delays Aphid Feeding.

The plant cell wall plays an important role in damage-associated molecular pattern-induced resistance to pathogens and herbivorous insects. Our current understanding of cell wall-mediated resistance is largely based on the degree of pectin methylesterification. However, little is known about the role of pectin acetylesterification in plant immunity. This study describes how one pectin-modifying enzyme, PECTIN ACETYLESTERASE 9 (PAE9), affects the Arabidopsis (Arabidopsis thaliana) transcriptome, secondary metabolome, and aphid performance. Electro-penetration graphs showed that Myzus persicae aphids established phloem feeding earlier on pae9 mutants. Whole-genome transcriptome analysis revealed a set of 56 differentially expressed genes (DEGs) between uninfested pae9-2 mutants and wild-type plants. The majority of the DEGs were enriched for biotic stress responses and down-regulated in the pae9-2 mutant, including PAD3 and IGMT2, involved in camalexin and indole glucosinolate biosynthesis, respectively. Relative quantification of more than 100 secondary metabolites revealed decreased levels of several compounds, including camalexin and oxylipins, in two independent pae9 mutants. In addition, absolute quantification of phytohormones showed that jasmonic acid (JA), jasmonoyl-Ile, salicylic acid, abscisic acid, and indole-3-acetic acid were compromised due to PAE9 loss of function. After aphid infestation, however, pae9 mutants increased their levels of camalexin, glucosinolates, and JA, and no long-term effects were observed on aphid fitness. Overall, these data show that PAE9 is required for constitutive up-regulation of defense-related compounds, but that it is not required for aphid-induced defenses. The signatures of phenolic antioxidants, phytoprostanes, and oxidative stress-related transcripts indicate that the processes underlying PAE9 activity involve oxidation-reduction reactions.

The chromatin-modifying protein HUB2 is involved in the regulation of lignin composition in xylem vessels.

PIRIN2 (PRN2) was earlier reported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis thaliana. In the present study, we report yeast two-hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by ß-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis-GC/MS. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier transform infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analysis of the hub2 prn2 double mutant, support the antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic gene FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localized in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin-promoting function of HUB2 is antagonized by PRN2 for the benefit of the guaiacyl (G)-type lignin enrichment of the neighbouring xylem vessel elements.

AspWood: High-Spatial-Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula.

Trees represent the largest terrestrial carbon sink and a renewable source of ligno-cellulose. There is significant scope for yield and quality improvement in these largely undomesticated species, and efforts to engineer elite varieties will benefit from improved understanding of the transcriptional network underlying cambial growth and wood formation. We generated high-spatial-resolution RNA sequencing data spanning the secondary phloem, vascular cambium, and wood-forming tissues of Populus tremula The transcriptome comprised 28,294 expressed, annotated genes, 78 novel protein-coding genes, and 567 putative long intergenic noncoding RNAs. Most paralogs originating from the Salicaceae whole-genome duplication had diverged expression, with the exception of those highly expressed during secondary cell wall deposition. Coexpression network analyses revealed that regulation of the transcriptome underlying cambial growth and wood formation comprises numerous modules forming a continuum of active processes across the tissues. A comparative analysis revealed that a majority of these modules are conserved in Picea abies The high spatial resolution of our data enabled identification of novel roles for characterized genes involved in xylan and cellulose biosynthesis, regulators of xylem vessel and fiber differentiation and lignification. An associated web resource (AspWood, http://aspwood.popgenie.org) provides interactive tools for exploring the expression profiles and coexpression network.

Transcriptome analysis of shade avoidance and shade tolerance in conifers.

MAIN CONCLUSION: Gymnosperms respond differently to light intensity and R:FR; although some aspects of shade response appear conserved, yet underlying mechanisms seem to be diverse in gymnosperms as compared to angiosperms. Shade avoidance syndrome (SAS) is well-characterized in the shade intolerant model species Arabidopsis thaliana whereas much less is known about shade tolerance response (STR), yet regulation of SAS and STR with reference to conifers remains poorly understood. We conducted a comparative study of two conifer species with contrasting responses to shade, Scots pine (shade-intolerant) and Norway spruce (shade-tolerant), with the aim to understand mechanisms behind SAS and STR in conifers. Pine and spruce seedlings were grown under controlled light and shade conditions, and hypocotyl and seedling elongation following different light treatments were determined in both species as indicators of shade responses. Red to far-red light ratio (R:FR) was shown to trigger the shade response in Norway spruce. In Scots pine, we observed an interaction between R:FR and light intensity. RNA sequencing (RNA-Seq) data revealed that SAS and STR responses included changes in expression of genes involved primarily in hormone signalling and pigment biosynthesis. From the RNA-Seq analysis, we propose that although some aspects of shade response appear to be conserved in angiosperms and gymnosperms, yet the underlying mechanisms may be different in gymnosperms that warrants further research.

An AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition.

Differentiation of xylem elements involves cell expansion, secondary cell wall (SCW) deposition and programmed cell death. Transitions between these phases require strict spatiotemporal control. The function of Populus ERF139 (Potri.013G101100) in xylem differentiation was characterized in transgenic overexpression and dominant repressor lines of ERF139 in hybrid aspen (Populus tremula × tremuloides). Xylem properties, SCW chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, Fourier transform-infrared spectroscopy, pyrolysis-GC/MS, wet chemistry methods and RNA sequencing. Opposite phenotypes were observed in the secondary xylem vessel sizes and SCW chemistry in the two different types of transgenic trees, supporting the function of ERF139 in suppressing the radial expansion of vessel elements and stimulating accumulation of guaiacyl-type lignin and possibly also xylan. Comparative transcriptomics identified genes related to SCW biosynthesis (LAC5, LBD15, MYB86) and salt and drought stress-responsive genes (ANAC002, ABA1) as potential direct targets of ERF139. The phenotypes of the transgenic trees and the stem expression profiles of ERF139 potential target genes support the role of ERF139 as a transcriptional regulator of xylem cell expansion and SCW formation, possibly in response to osmotic changes of the cells.