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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.

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.

Integration of environmental and endogenous cues triggers floral induction at the optimal time during the plant life cycle. Flowering is a tightly regulated process, which involves an intricated genetic network, as expected for a process crucial for plant fitness and survival. Individual metabolites are known to contribute to the determination of flowering time, including carbohydrates and hormones. However, a global analysis of metabolic changes associated with flowering was still lacking. We performed a metabolomic study to characterize global metabolic changes associated with photoperiodic floral induction. By using an inducible system, with the CONSTANS (CO) promoter driving the expression of CO fused to the rat glucocorticoid receptor (CO::GR), we induce flowering and identify metabolites that increase or decrease in leaves and apices during floral induction. Combining metabolomic with transcriptomic data, we identify that raffinose metabolism was altered in apices that are induced to flower. Loss-of-function mutants affecting RAFFINOSE SYNTHASE 5 (RS5), a key enzyme of the raffinose metabolism, show an early flowering phenotype. Also, RS5 expression changes during floral transition, suggesting a role for raffinose catabolism on the release of simple sugars at the apex. We propose that variation on the differential accumulation of raffinose and mono- and disaccharides during floral transition contributes to the induction of floral transition, by influencing expression of THEHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), which affect expression of the florigen FLOWERING LOCUS T (FT).

European aspen (Populus tremula L.) undergoes a coordinated senescence program during autumn; however, it is not known what exactly triggers it. To identify the cellular program leading to senescence, we utilized natural variation among Swedish aspen genotypes in a common garden to study senescence timing and the underlying changes in leaf phytohormone and transcriptome profiles. Apart from the patterns of major transcriptional cascade that was similar between the genotypes and closely associated with cytokinin and auxin metabolite levels and gradually decreasing air temperature during autumn, we detected patterns that consistently preceded or coincided with senescence onset in individual genotypes. Another cascade seemed to respond to short-term changes in weather conditions that re-wired the transcriptional network; the up-regulation of genes related to ethylene and abiotic stress, programmed cell death and translation occurred first in the early-senescing genotypes and later in the late one. Network analyses displayed a connection between the two cascades, metabolic stress and immunity responses mediated by salicylic acid (SA)-signalling pathway that was repressed along with SA levels at senescence onset. We propose that autumn senescence in aspen trees is affected by environmental variation that evokes stress and the timing is fine-tuned by their stress tolerance mechanism.One sentence summarySalicylic acid signalling pathway fine-tunes the timing of senescence onset under challenging environmental conditions in autumn.

In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.(1) This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.(1-4) The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.(3,5) Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy(.3,5). In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle.

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.

Pools of arginine and ornithine generated during protein degradation can pace the progression of leaf senescence by affecting the TCA cycle, polyamine biosynthesis and the ethylene signaling pathway.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.

Secondary growth relies on precise and specialized transcriptional networks that determine cell division, differentiation, and maturation of xylem cells. We identify a novel role for the ethylene induced Populus ETHYLENE RESPONSE FACTOR ERF85 (Potri.015G023200) in balancing xylem cell expansion and secondary cell wall (SCW) formation in hybrid aspen (Populus tremula x tremuloides). Expression of ERF85 is high in phloem and cambium cells and during expansion of xylem cells, while it is low in maturing xylem tissue. Extending ERF85 expression into SCW forming zones of woody tissues through ectopic expression reduced wood density and SCW thickness of xylem fibers but increased fiber diameter. Xylem transcriptomes from the transgenic trees revealed transcriptional induction of genes involved in cell expansion, translation and growth. Expression of genes associated with plant vascular development and biosynthesis of SCW chemical components such as xylan and lignin, was downregulated in the transgenic trees. Our results suggest that ERF85 activates genes related with xylem cell expansion, while preventing transcriptional activation of genes related to SCW formation. The importance of precise spatial expression of ERF85 during wood development together with the observed phenotypes in response to ectopic ERF85 expression suggests that ERF85 functions as a switch between different phases of xylem differentiation during wood formation.

Meiotic recombination is a ubiquitous function of sexual reproduction throughout eukaryotes. Here we report that recombination is extremely suppressed during meiosis in the yeast speciesSaccharomycodes ludwigii. DNA double-strand break formation, processing and repair are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers. We propose thatSd. ludwigiihas followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent fertilization within the meiotic tetrad with the absence of meiotic recombination.

Background Translation is a tightly regulated process, controlling the rate of protein synthesis in cells. Ribosome sequencing (Ribo-Seq) is a recently developed tool for studying actively translated mRNA and can thus directly address translational regulation. Ribo-Seq libraries need to be sequenced to a great depth due to high contamination by rRNA and other contaminating nucleic acid fragments. Deep sequencing is expensive, and it generates large volumes of data, making data analysis complicated and time consuming. Methods and results Here we developed a platform for Ribo-Seq library construction and data analysis to enable rapid quality assessment of Ribo-Seq libraries with the help of a small-scale sequencer. Our data show that several qualitative features of a Ribo-Seq library, such as read length distribution, P-site distribution, reading frame and triplet periodicity, can be effectively evaluated using only the data generated by a benchtop sequencer with a very limited number of reads. Conclusion Our pipeline enables rapid evaluation of Ribo-Seq libraries, opening up possibilities for optimization of Ribo-Seq library construction from difficult samples, and leading to better decision making prior to more costly deep sequencing.

In eukaryotes, TOR (Target Of Rapamycin) is a conserved regulator of growth that integrates both endogenous and exogenous signals. These signals include the internal nutritional status, and in plants, TOR has been shown to be regulated by carbon, nitrogen and sulfur availability. In this study, we show that in Arabidopsis the TOR pathway also integrates phosphorus availability to actively modulate the cell cycle, which in turn regulates the intracellular content of amino acids and organic acids. We observed a substantial overlap between the phenotypic, metabolic and transcriptomic responses of TOR inactivation and phosphorus starvation in Arabidopsis cell culture. Although phosphorus availability modulates TOR activity, changes in the levels of TOR activity do not alter the expression of marker genes for phosphorus status. These data prompted us to place the sensing of phosphorus availability upstream of the modulation of TOR activity which, in turn, regulates the cell cycle and primary metabolism to adjust plant growth in plants.

Microbial communities are major players in carbon and nitrogen cycling globally and are of particular importance for plant communities in the nutrient poor soils of boreal forests. Especially relevant are the fungal communities in the soil that interact with the plants in multiple ways, indirectly through their pivotal role in the breakdown of organic matter and, more directly, through mycorrhizal symbiosis with plant roots. Large-scale disturbances of these complex microbial communities can lead to shifts in soil carbon storage with unknown and global-scale long-term consequences. To understand the dynamics of these communities and their relationship to associated plants in response to climate change and anthropogenic influence, we need a better understanding of how modern “omics” methods can help us to understand compositional and functional shifts of these microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from environmental samples. In contrast, currently phylogenetic marker gene amplicon sequencing data is generally used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in RNA-Seq transcriptomic data from matched samples. Here we describe fungal communities using both RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from mature stands of the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient deficient) and nutrient enriched plots at the Flakaliden forest research site in boreal northern Sweden. We created an assembly-based, reproducible and hardware agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. We show that the community structure indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying limitations imposed by current database coverage. Furthermore, we show examples to demonstrate how metatranscriptomics data additionally provides biologically informative functional insight at the community and individual species level. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response and effect both between host plants and their associated microbial communities, and among the members of microbial communities in environmental samples in general.

The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus Cortinarius. IMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.

BackgroundIntermixing of genomes through meiotic reassortment and recombination of homologous chromosomes is a unifying theme of sexual reproduction in eukaryotic organisms and is considered crucial for their adaptive evolution. Previous studies of the budding yeast speciesSaccharomycodes ludwigiisuggested that meiotic crossing over might be absent from its sexual life cycle, which is predominated by fertilization within the meiotic tetrad.ResultsWe demonstrate that recombination is extremely suppressed during meiosis inSd. ludwigii. DNA double-strand break formation by the conserved transesterase Spo11, processing and repair involving interhomolog interactions are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers in its genome. A strong AT bias of spontaneous mutations and the absence of recombination are likely responsible for its unusually low genomic GC level.ConclusionsSd. ludwigiihas followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent mating between products of the same meiotic event with the extreme suppression of meiotic recombination. This life style ensures preservation of heterozygosity throughout its genome and may enable the species to adapt to its environment and survive with only minimal levels of rare meiotic recombination. We proposeSd. ludwigiias an excellent natural forum for the study of genome evolution and recombination rates.

SummaryBoreal 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 needles as a resource to understand needle development and acclimation processes throughout the year. In young, growing needles (May 15 – 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.Significance statementThe development of Norway spruce needles and their annual cycle of biochemical activities (photosynthesis in the summer, adaptation and survival of the harsh boreal winter) is not well understood. We use deep RNA sequencing to profile the transcriptome over the season, and show how the dataset could be used to give information about “what needles do” over the year, and are here making the dataset available for the scientific community.Graphical abstract

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.

International audience; 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.

Seasonal cues influence several aspects of the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. We investigated seasonal changes in cambial activity, secondary cell wall formation, and tracheid cell death in woody tissues of Norway spruce (Picea abies) throughout one seasonal cycle. RNA sequencing was performed simultaneously in both the xylem and cambium/phloem tissues of the stem. Principal component analysis revealed gradual shifts in the transcriptomes that followed a chronological order throughout the season. A notable remodeling of the transcriptome was observed in the winter, with many genes having maximal expression during the coldest months of the year. A highly coexpressed set of monolignol biosynthesis genes showed high expression during the period of secondary cell wall formation as well as a second peak in midwinter. This midwinter peak in expression did not trigger lignin deposition, as determined by pyrolysis-gas chromatography/mass spectrometry. Coexpression consensus network analyses suggested the involvement of transcription factors belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES and MYELOBLASTOSIS-HOMEOBOX families in the seasonal control of secondary cell wall formation of tracheids. Interestingly, the lifetime of the latewood tracheids stretched beyond the winter dormancy period, correlating with a lack of cell death-related gene expression. Our transcriptomic analyses combined with phylogenetic and microscopic analyses also identified the cellulose and lignin biosynthetic genes and putative regulators for latewood formation and tracheid cell death in Norway spruce, providing a toolbox for further physiological and functional assays of these important phase transitions.

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, ...

Background Drought and salinity are two of the main challenges in agriculture. In many areas, crop production needs solutions to adapt the grown species to the increasing salinity. Research on physiological and molecular responses activated by salinity in plants is needed to elucidate mechanisms of salinity tolerance. Transcriptome profiling (RNA-Seq) is a powerful tool to study the transcriptomic profile of genotypes under stress conditions. In temperate fruit tree species, tree grafting on salinity tolerant rootstocks is a common method to compensate for the cultivar saline sensitivity. Persimmon species have different levels of tolerance to salinity, knowledge of this variability provides the basics for development of salt tolerant rootstocks.Results In this study, we conducted a physiological and transcriptomic profiling of roots and leaves in tolerant and sensitive plants of persimmon rootstock, Diospyros lotus, grown under saline and control conditions. Results from characterization of the physiological responses along with gene expression changes in roots and leaves allowed identifying several salt-tolerance mechanisms related to Ion transport and thermospermine synthesis. Differences were observed in putative H+/ATPases that allow transmembrane ionic transport and Chloride channel protein-like genes. Furthermore, an overexpression of thermospermine synthase found in the roots of tolerant plants may indicate that alterations in root architecture could act as an additional mechanism of response to salt stress. Conclusions Results indicate that D. lotus presents a genetic variability for salt tolerance trait related to the regulation of chloride transport, transmembrane electrochemical potential and thermospermine root synthesis. The study provides knowledge on mechanism of salt stress tolerance in persimmon for further breeding of tolerant persimmon rootstocks.

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 β-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.

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pre-genome, qualitative methods. To help address these limitations, we combined machine learning andin silicoenrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae:Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats inP. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about a third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly-studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.

Differentiation of xylem elements involves cell expansion, secondary cell wall 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 x tremuloides). Xylem properties, secondary cell wall (SCW) chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, FT-IR, 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. This article is protected by copyright. All rights reserved.

The genome assembly of the European aspen Populus tremula proved difficult for a short-read based strategy due to high genomic variation. As a consequence, the fragmented sequence is impeding studies that benefit from highly contiguous data, particularly genome-wide association studies (GWAS) and comparative genomics. Here we present an updated assembly based on long-read sequences, optical mapping and genetic mapping. This assembly - henceforth referred to as Potra V2 - is assembled into 19 contiguous chromosomes which provides a powerful tool for future association studies. The genome sequence and any feature files are available from the PopGenIE resource.

Seed maturation is an important plant developmental process that follows embryo development. It is associated with a series of physiological changes such as the establishment of desiccation tolerance, seed longevity and seed dormancy. However, the translational dynamics associated with seed maturation, especially its connection with seed germination remains largely elusive. Here transcriptome and translatome profiling were performed during seed maturation. During seed maturation we observed a gradual disappearance of polysomes and a relative increase of monosomes, indicating a gradual reduction of global translation. Comparing the levels of polysomal associated mRNAs with total mRNA levels showed that thousands of genes are translationally regulated at early sates of maturation, as judged by dramatic changes in polysomal occupancy. By including previous published data from germination and seedling establishment, a translational regulatory network: SeedTransNet was constructed. Network analysis identified hundreds of gene modules with distinct functions and transcript sequence features indicating the existence of separate translational regulatory circuits possibly acting through specific regulatory elements. The regulatory potential of one such element was confirmed in vivo. The network identified several seed maturation associated genes as central nodes, and we could confirm the importance of many of these hub genes with a maturation associated seed phenotype by mutant analysis. One of the identified regulators an AWPM19 family protein PM19-Like1 (PM19L1) was shown to regulate seed dormancy and longevity. This putative RBP also affects the transitional regulation of one its, by the SeedTransNet identified, target mRNAs. Our data shows the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

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. Transcriptomics combined with single-cell metabolomics give new information on the role of rays in lignification of developing xylem in Norway spruce.