Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

N-glycoproteins in Plant Cell Walls: A Survey.

Cell walls are an extracellular compartment specific to plant cells, which are not found in animal cells. Their composition varies between cell types, plant species, and physiological states. They are composed of a great diversity of polymers, i.e., polysaccharides, proteins, and lignins. Cell wall proteins (CWPs) are major players involved in the plasticity of cell walls which support cell growth and differentiation, as well as adaptation to environmental changes. In order to reach the extracellular space, CWPs are transported through the secretory pathway where they may undergo post-translational modifications, including N-glycosylations on the Asn residues in specific motifs (Asn-X-Ser/Thr-X, with X≠Pro). This review aims at providing a survey of the present knowledge related to cell wall N-glycoproteins with (i) an overview of the experimental workflows, (ii) a selection of relevant articles dedicated to N-glycoproteomics, (iii) a description of the diversity of N-glycans, and (iv) a focus on the importance of N-glycans for CWP structure and/or function.

Plant Cell Wall Proteomes: The Core of Conserved Protein Families and the Case of Non-Canonical Proteins.

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways.

Characterization of plant microRNA-encoded peptides (miPEPs) reveals molecular mechanisms from the translation to activity and specificity.

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

A Comprehensive Assessment of the Secretome Responsible for Host Adaptation of the Legume Root Pathogen Aphanomyces euteiches.

The soil-borne oomycete pathogen Aphanomyces euteiches causes devastating root rot diseases in legumes such as pea and alfalfa. The different pathotypes of A. euteiches have been shown to exhibit differential quantitative virulence, but the molecular basis of host adaptation has not yet been clarified. Here, we re-sequenced a pea field reference strain of A. euteiches ATCC201684 with PacBio long-reads and took advantage of the technology to generate the mitochondrial genome. We identified that the secretome of A. euteiches is characterized by a large portfolio of secreted proteases and carbohydrate-active enzymes (CAZymes). We performed Illumina sequencing of four strains of A. euteiches with contrasted specificity to pea or alfalfa and found in different geographical areas. Comparative analysis showed that the core secretome is largely represented by CAZymes and proteases. The specific secretome is mainly composed of a large set of small, secreted proteins (SSP) without any predicted functional domain, suggesting that the legume preference of the pathogen is probably associated with unknown functions. This study forms the basis for further investigations into the mechanisms of interaction of A. euteiches with legumes.

The Same against Many: AtCML8, a Ca2+ Sensor Acting as a Positive Regulator of Defense Responses against Several Plant Pathogens.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.

Physiological and Biochemical Traits of Two Major Arabidopsis Accessions, Col-0 and Ws, Under Salinity.

Salinity affects plant growth and development as shown with the glycophyte model plant, Arabidopsis thaliana (Arabidopsis). Two Arabidopsis accessions, Wassilewskija (Ws) and Columbia (Col-0), are widely used to generate mutants available from various Arabidopsis seed resources. However, these two ecotypes are known to be salt-sensitive with different degrees of tolerance. In our study, 3-week-old Col-0 and Ws plants were treated with and without 150 mM NaCl for 48, 72, or 96 h, and several physiological and biochemical traits were characterized on shoots to identify any specific traits in their tolerance to salinity. Before salt treatment was carried out, a different phenotype was observed between Col-0 and Ws, whose main inflorescence stem became elongated in contrast to Col-0, which only displayed rosette leaves. Our results showed that Col-0 and Ws were both affected by salt stress with limited growth associated with a reduction in nutrient uptake, a degradation of photosynthetic pigments, an increase in protein degradation, as well as showing changes in carbohydrate metabolism and cell wall composition. These traits were often more pronounced in Col-0 and occurred usually earlier than in Ws. Tandem Mass Tags quantitative proteomics data correlated well with the physiological and biochemical results. The Col-0 response to salt stress was specifically characterized by a greater accumulation of osmoprotectants such as anthocyanin, galactinol, and raffinose; a lower reactive oxygen detoxification capacity; and a transient reduction in galacturonic acid content. Pectin degradation was associated with an overaccumulation of the wall-associated kinase 1, WAK1, which plays a role in cell wall integrity (CWI) upon salt stress exposure. Under control conditions, Ws produced more antioxidant enzymes than Col-0. Fewer specific changes occurred in Ws in response to salt stress apart from a higher number of different fascilin-like arabinogalactan proteins and a greater abundance of expansin-like proteins, which could participate in CWI. Altogether, these data indicate that Col-0 and Ws trigger similar mechanisms to cope with salt stress, and specific changes are more likely related to the developmental stage than to their respective genetic background.

Comparison of mass spectrometry data and bioinformatics predictions to assess the bona fide localization of proteins identified in cell wall proteomics studies.

Plant cell walls have complex architectures made of polysaccharides among which cellulose, hemicelluloses, pectins and cell wall proteins (CWPs). Some CWPs are anchored in the plasma membrane through a glycosylphosphatidylinositol (GPI)-anchor. The secretion pathway is the classical route to reach the extracellular space. Based on experimental data, a canonical signal peptide (SP) has been defined, and bioinformatics tools allowing the prediction of the sub-cellular localization of proteins have been designed. In the same way, the presence of GPI-anchor attachment sites can be predicted using bioinformatics programs. This article aims at comparing the bioinformatics predictions of the sub-cellular localization of proteins assumed to be CWPs to mass spectrometry (MS) data. The sub-cellular localization of a few CWPs exhibiting particular features has been checked by cell biology approaches. Although the prediction of SP length is confirmed in most cases, it is less conclusive for GPI-anchors. Three main observations were done: (i) the variability observed at the N-terminus of a few mature CWPs could play a role in the regulation of their biological activity; (ii) one protein was shown to have a double sub-cellular localization in the cell wall and the chloroplasts; and (iii) peptides were found to be located at the C-terminus of several CWPs previously identified in GPI-anchored proteomes, thus raising the issue of their actual anchoring to the plasma membrane.

A Tandem Mass Tags (TMTs) labeling approach highlights differences between the shoot proteome of two Arabidopsis thaliana ecotypes, Col-0 and Ws.

Arabidopsis has become a powerful model to study morphogenesis, plant growth, development but also plant response to environmental conditions. Over 1000 Arabidopsis genomes are available and show natural genetic variations. Among them, the main reference accessions Wassilewskija (Ws) and Columbia (Col-0), originally growing at contrasted altitudes and temperatures, are widely studied, but data contributing to their molecular phenotyping are still scarce. A global quantitative proteomics approach using isobaric stable isotope labeling (Tandem Mass Tags, TMT) was performed on Ws and Col-0. Plants have been hydroponically grown at 16 h/8 h (light/dark cycle) at 23°C day/19°C night for three weeks. A TMT labeling of the proteins extracted from their shoots has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several proteins families possibly involved in the differential responses observed for Ws and Col-0 during plant development and upon environmental changes. In particular, Ws and Col-0 mainly differ in photosynthesis, cell wall-related proteins, plant defense/stress, ROS scavenging enzymes/redox homeostasis and DNA/RNA binding/transcription/translation/protein folding.

Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8.

BACKGROUND: Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. RESULTS: We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. CONCLUSION: Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

Cell wall proteomic datasets of stems and leaves of Brachypodium distachyon.

This article provides experimental data describing the cell wall protein profiles of stems and leaves of Brachypodium distachyon at two different stages of development. The cell wall proteomics data have been obtained from (i) stem internodes at young and mature stages of development, and (ii) leaves at young and mature stages of development. The proteins have been extracted from purified cell walls using buffers containing calcium chloride (0.2 M) or lithium chloride (2 M). They have been identified by LC-MS/MS and bioinformatics. These data allow deepening our knowledge of these cell wall proteomes. They are a valuable resource for people interested in plant cell wall biology to understand the roles of cell wall proteins during the growth of vegetative organs.

Global Evolutionary Analysis of 11 Gene Families Part of Reactive Oxygen Species (ROS) Gene Network in Four Eucalyptus Species.

Eucalyptus is a worldwide hard-wood species which increasingly focused on. To adapt to various biotic and abiotic stresses, Eucalyptus have evolved complex mechanisms, increasing the cellular concentration of reactive oxygen species (ROS) by numerous ROS controlling enzymes. To better analyse the ROS gene network and discuss the differences between four Eucalyptus species, ROS gene network including 11 proteins families (1CysPrx, 2CysPrx, APx, APx-R, CIII Prx, Diox, GPx, Kat, PrxII, PrxQ and Rboh) were annotated and compared in an expert and exhaustive manner from the genomic data available from E. camaldulensis, E. globulus, E. grandis, and E. gunnii. In addition, a specific sequencing strategy was performed in order to determine if the missed sequences in at least one organism are the results of gain/loss events or only sequencing gaps. We observed that the automatic annotation applied to multigenic families is the source of miss-annotation. Base on the family size, the 11 families can be categorized into duplicated gene families (CIII Prx, Kat, 1CysPrx, and GPx), which contain a lot of gene duplication events and non-duplicated families (APx, APx-R, Rboh, DiOx, 2CysPrx, PrxII, and PrxQ). The gene family sizes are much larger in Eucalyptus than most of other angiosperms due to recent gene duplications, which could give higher adaptability to environmental changes and stresses. The cross-species comparative analysis shows gene gain and loss events during the evolutionary process. The 11 families possess different expression patterns, while in the Eucalyptus genus, the ROS families present similar expression patterns. Overall, the comparative analysis might be a good criterion to evaluate the adaptation of different species with different characters, but only if data mining is as exhaustive as possible. It is also a good indicator to explore the evolutionary process.

The Cell Wall PAC (Proline-Rich, Arabinogalactan Proteins, Conserved Cysteines) Domain-Proteins Are Conserved in the Green Lineage.

Plant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain (Proline-rich, Arabinogalactan proteins, conserved Cysteines) in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of ß-sheets is assumed to ensure the correct folding of the PAC domain as a ß-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymers.

Advanced Cataloging of Lysine-63 Polyubiquitin Networks by Genomic, Interactome, and Sensor-Based Proteomic Analyses.

The lack of resolution when studying the many different ubiquitin chain types found in eukaryotic cells has been a major hurdle to our understanding of their specific roles. We currently have very little insight into the cellular and physiological functions of Lys-63 (K63)-linked ubiquitin chains, although they are the second most abundant forms of ubiquitin in plant cells. To overcome this problem, we developed several large-scale approaches to characterize (1) the E2-E3 ubiquitination machinery driving K63-linked ubiquitin chain formation and (2) K63 polyubiquitination targets to provide a comprehensive picture of K63 polyubiquitin networks in Arabidopsis (Arabidopsis thaliana). Our work identified the ubiquitin-conjugating enzymes (E2s) UBC35/36 as the major drivers of K63 polyubiquitin chain formation and highlights the major role of these proteins in plant growth and development. Interactome approaches allowed us to identify many proteins that interact with the K63 polyubiquitination-dedicated E2s UBC35/36 and their cognate E2 variants, including more than a dozen E3 ligases and their putative targets. In parallel, we improved the in vivo detection of proteins decorated with K63-linked ubiquitin chains by sensor-based proteomics, yielding important insights into the roles of K63 polyubiquitination in plant cells. This work strongly increases our understanding of K63 polyubiquitination networks and functions in plants.

Long-Read Genome Sequence of the Sugar Beet Rhizosphere Mycoparasite Pythium oligandrum.

Pythium oligandrum is a soil born free living oomycete able to parasitize fungi and oomycetes prey, including important plant and animals pathogens. Pythium oligandrum can colonize endophytically the root tissues of diverse plants where it induces plant defenses. Here we report the first long-read genome sequencing of a P. oligandrum strain sequenced by PacBio technology. Sequencing of genomic DNA loaded onto six SMRT cells permitted the acquisition of 913,728 total reads resulting in 112X genome coverage. The assembly and polishing of the genome sequence yielded180 contigs (N50 = 1.3 Mb; L50 = 12). The size of the genome assembly is 41.9 Mb with a longest contig of 2.7 Mb and 15,007 predicted protein-coding genes among which 95.25% were supported by RNAseq data, thus constituting a new Pythium genome reference. This data will facilitate genomic comparisons of Pythium species that are commensal, beneficial or pathogenic on plant, or parasitic on fungi and oomycete to identify key genetic determinants underpinning their diverse lifestyles. In addition comparison with plant pathogenic or zoopathogenic species will illuminate genomic adaptations for pathogenesis toward widely diverse hosts.

Phenotypic Trait Variation as a Response to Altitude-Related Constraints in Arabidopsis Populations.

Natural variations help in identifying genetic mechanisms of morphologically and developmentally complex traits. Mountainous habitats provide an altitudinal gradient where one species encounters different abiotic conditions. We report the study of 341 individuals of Arabidopsis thaliana derived from 30 natural populations not belonging to the 1001 genomes, collected at increasing altitudes, between 200 and 1800 m in the Pyrenees. Class III peroxidases and ribosomal RNA sequences were used as markers to determine the putative genetic relationships among these populations along their altitudinal gradient. Using Bayesian-based statistics and phylogenetic analyses, these Pyrenean populations appear with significant divergence from the other regional accessions from 1001 genome (i.e., from north Spain or south France). Individuals of these populations exhibited varying phenotypic changes, when grown at sub-optimal temperature (22 vs. 15°C). These phenotypic variations under controlled conditions reflected intraspecific morphological variations. This study could bring new information regarding the west European population structure of A. thaliana and its phenotypic variations at different temperatures. The integrative analysis combining genetic, phenotypic variation and environmental datasets is used to analyze the acclimation of population in response to temperature changes. Regarding their geographical proximity and environmental diversity, these populations represent a tool of choice for studying plant response to temperature variation. HIGHLIGHTS: -Studying the natural diversity of A. thaliana in the Pyrenees mountains helps to understand European population structure and to evaluate the phenotypic trait variation in response to climate change.

A systems biology view of wood formation in Eucalyptus grandis trees submitted to different potassium and water regimes.

Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Functional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological processes and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further confirmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits.

Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.