Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination.

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of a ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. While extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it is so far unknown how these translate to quantitative system behaviour in vivo, a problem that is confounded by large NAP gene families in most species. Here we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knock-in fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during developmental progression. Thus, quantitative analysis of the entire NAP allowed us to identify ARF degradation and stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

RAF-like protein kinases mediate a deeply conserved, rapid auxin response.

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

Assessment of infant outgrowth of cow's milk allergy in relation to the faecal microbiome and metaproteome.

Previous studies provide evidence for an association between modifications of the gut microbiota in early life and the development of food allergies. We studied the faecal microbiota composition (16S rRNA gene amplicon sequencing) and faecal microbiome functionality (metaproteomics) in a cohort of 40 infants diagnosed with cow's milk allergy (CMA) when entering the study. Some of the infants showed outgrowth of CMA after 12 months, while others did not. Faecal microbiota composition of infants was analysed directly after CMA diagnosis (baseline) as well as 6 and 12 months after entering the study. The aim was to gain insight on gut microbiome parameters in relation to outgrowth of CMA. The results of this study show that microbiome differences related to outgrowth of CMA can be mainly identified at the taxonomic level of the 16S rRNA gene, and to a lesser extent at the protein-based microbial taxonomy and functional protein level. At the 16S rRNA gene level outgrowth of CMA is characterized by lower relative abundance of Lachnospiraceae at baseline and lower Bacteroidaceae at visit 12 months.

The birth of a giant: evolutionary insights into the origin of auxin responses in plants.

The plant signaling molecule auxin is present in multiple kingdoms of life. Since its discovery, a century of research has been focused on its action as a phytohormone. In land plants, auxin regulates growth and development through transcriptional and non-transcriptional programs. Some of the molecular mechanisms underlying these responses are well understood, mainly in Arabidopsis. Recently, the availability of genomic and transcriptomic data of green lineages, together with phylogenetic inference, has provided the basis to reconstruct the evolutionary history of some components involved in auxin biology. In this review, we follow the evolutionary trajectory that allowed auxin to become the "giant" of plant biology by focusing on bryophytes and streptophyte algae. We consider auxin biosynthesis, transport, physiological, and molecular responses, as well as evidence supporting the role of auxin as a chemical messenger for communication within ecosystems. Finally, we emphasize that functional validation of predicted orthologs will shed light on the conserved properties of auxin biology among streptophytes.

A long look at short prokaryotic Argonautes.

Argonaute proteins (Agos) use small 15-30 nucleotide-long guides to bind and/or cleave complementary target nucleic acids. Eukaryotic Agos mediate RNA-guided RNA silencing, while 'long' prokaryotic Agos (pAgos) use RNA or DNA guides to interfere with invading plasmid and viral DNA. Here, we review the function and mechanisms of truncated and highly divergent 'short' pAgos, which, until recently, remained functionally uncharacterized. Short pAgos have retained the Middle (MID) and P-element-Induced Wimpy Testis (PIWI) domains important for guide-mediated target binding, but lack the ability to cleave their targets. Instead, emerging insights reveal that various short pAgos interact with distinct accessory 'effector' enzymes. Upon guide-mediated detection of invading DNA by short pAgos, their associated effector enzymes kill the host cell and, consequentially, prevent spread of the invader.

Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA.

Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

Pole position: How plant cells polarize along the axes.

Having a sense of direction is a fundamental cellular trait that can determine cell shape, division orientation, or function, and ultimately the formation of a functional, multicellular body. Cells acquire and integrate directional information by establishing discrete subcellular domains along an axis with distinct molecular profiles, a process known as cell polarization. Insight into the principles and mechanisms underlying cell polarity has been propelled by decades of extensive research mostly in yeast and animal models. Our understanding of cell polarity establishment in plants, which lack most of the regulatory molecules identified in other eukaryotes, is more limited, but significant progress has been made in recent years. In this review, we explore how plant cells coordinately establish stable polarity axes aligned with the organ axes, highlighting similarities in the molecular logic used to polarize both plant and animal cells. We propose a classification system for plant cell polarity events and nomenclature guidelines. Finally, we provide a deep phylogenetic analysis of polar proteins and discuss the evolution of polarity machineries in plants.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

Deep Evolutionary History of the Phox and Bem1 (PB1) Domain Across Eukaryotes.

Protein oligomerization is a fundamental process to build complex functional modules. Domains that facilitate the oligomerization process are diverse and widespread in nature across all kingdoms of life. One such domain is the Phox and Bem1 (PB1) domain, which is functionally well-studied in the animal kingdom. However, beyond animals, neither the origin nor the evolutionary patterns of PB1-containing proteins are understood. While PB1 domain proteins have been found in other kingdoms including plants, it is unclear how these relate to animal PB1 proteins. To address this question, we utilized large transcriptome datasets along with the proteomes of a broad range of species. We discovered eight PB1 domain-containing protein families in plants, along with four each in Protozoa and Fungi and three families in Chromista. Studying the deep evolutionary history of PB1 domains throughout eukaryotes revealed the presence of at least two, but likely three, ancestral PB1 copies in the Last Eukaryotic Common Ancestor (LECA). These three ancestral copies gave rise to multiple orthologues later in evolution. Analyzing the sequence and secondary structure properties of plant PB1 domains from all the eight families showed their common ubiquitin ß-grasp fold, despite poor sequence identity. Tertiary structural models of these plant PB1 families, combined with Random Forest based classification, indicated family-specific differences attributed to the length of PB1 domain and the proportion of ß-sheets. Thus, this study not only identifies novel PB1 families, but also provides an evolutionary basis to understand their diverse functional interactions.

Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts.

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

High-resolution and Deep Phylogenetic Reconstruction of Ancestral States from Large Transcriptomic Data Sets.

Phylogenetics is an important area of evolutionary biology that helps to understand the origin and divergence of genes, genomes and species. Building meaningful phylogenetic trees is needed for the accurate reconstruction of the past. To achieve a correct phylogenetic understanding of genes or proteins, reliable and robust methods are needed to construct meaningful trees. With the rapidly increasing availability of genome and transcriptome sequencing data, there is a need for efficient and accurate methodologies for ancestral state reconstruction. Currently available methods are mostly specific for certain gene families, and require substantial adaptation for their application to other gene families. Hence, a generalized framework is essential to utilize large transcriptome resources such as OneKP and MMETSP. Here, we have developed a flexible yet efficient method, based on core strengths such as emphasis on being inclusive in homolog selection, and defining orthologs based on multi-layered inferences. We illustrate how specific steps can be modified to fit the needs of any protein family under consideration. We also demonstrate the success of this protocol by studying and testing the orthologs in various gene families. Taken together, we present a protocol for reconstructing the ancestral states of various domains and proteins across multiple kingdoms of eukaryotes, using thousands of transcriptomes.

Evolution of vascular plants through redeployment of ancient developmental regulators.

Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.

Origin and evolution of the nuclear auxin response system.

The small signaling molecule auxin controls numerous developmental processes in land plants, acting mostly by regulating gene expression. Auxin response proteins are represented by large families of diverse functions, but neither their origin nor their evolution is understood. Here, we use a deep phylogenomics approach to reconstruct both the origin and the evolutionary trajectory of all nuclear auxin response protein families. We found that, while all subdomains are ancient, a complete auxin response mechanism is limited to land plants. Functional phylogenomics predicts defined steps in the evolution of response system properties, and comparative transcriptomics across six ancient lineages revealed how these innovations shaped a sophisticated response mechanism. Genetic analysis in a basal land plant revealed unexpected contributions of ancient non-canonical proteins in auxin response as well as auxin-unrelated function of core transcription factors. Our study provides a functional evolutionary framework for understanding diverse functions of the auxin signal.

Publisher Correction: Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo.

In the version of this Resource originally published, the author information was incorrect. Jos R. Wendrich should have had a present address: Department of Plant Biotechnology and Bioinformatics and VIB Center for Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Ghent, Belgium. Mark Boekschoten and Guido J. Hooiveld should have been affiliated to the Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands. In addition, the version of Supplementary Table 5 originally published with this Resource was not the intended final version and included inaccurate citations to the display items of the Resource, and the file format and extension did not match. These errors have now been corrected in all versions of the Resource.

Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo.

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.

Boosting LPMO-driven lignocellulose degradation by polyphenol oxidase-activated lignin building blocks.

BACKGROUND: Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. RESULTS: Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. CONCLUSIONS: The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.

Action of multiple intra-QTL genes concerted around a co-localized transcription factor underpins a large effect QTL.

Sub-QTLs and multiple intra-QTL genes are hypothesized to underpin large-effect QTLs. Known QTLs over gene families, biosynthetic pathways or certain traits represent functional gene-clusters of genes of the same gene ontology (GO). Gene-clusters containing genes of different GO have not been elaborated, except in silico as coexpressed genes within QTLs. Here we demonstrate the requirement of multiple intra-QTL genes for the full impact of QTL qDTY12.1 on rice yield under drought. Multiple evidences are presented for the need of the transcription factor 'no apical meristem' (OsNAM12.1) and its co-localized target genes of separate GO categories for qDTY12.1 function, raising a regulon-like model of genetic architecture. The molecular underpinnings of qDTY12.1 support its effectiveness in further improving a drought tolerant genotype and for its validity in multiple genotypes/ecosystems/environments. Resolving the combinatorial value of OsNAM12.1 with individual intra-QTL genes notwithstanding, identification and analyses of qDTY12.1has fast-tracked rice improvement towards food security.

Protein SUMOylation and plant abiotic stress signaling: in silico case study of rice RLKs, heat-shock and Ca(2+)-binding proteins.

Plants respond to stress conditions through early stress-response factors (ESRF), which serve the function of stress sensing and/or signal transduction. These mainly comprise qualitative and/or quantitative flux in the redox molecules, calcium ions (Ca(2+)), phosphatidic acid, hexose sugars and phytohormones. The role of resident proteins such as phytohormone receptors and G-proteins as first messengers under stress is well established. Yet, within the modern omics context, most of the stress response at the protein level is injudiciously attributed to substantial up- or down-regulation of expression measured at the RNA or protein level. Proteins such as kinases and transcription factors (TFs) that exhibit cascade effects are primary candidates for studies in plant stress tolerance. However, resident-protein post-translational modification (PTM), specifically in response to particular conditions such as stress, is a candidate for immediate and potent 'quick reaction force' (QRF) kind of effects. Stress-mediated SUMOylation of TFs and other proteins have been observed. SUMOylation can change the rate of activity, function or location of the modified protein. Early SUMOylation of resident proteins can act in the stress signal transduction or in adaptive response. Here, we consider brief background information on ESRFs to establish the crosstalk between these factors that impinge on PTMs. We then illustrate connections of protein SUMOylation to phytohormones and TFs. Finally, we present results of an in silico analysis of rice Receptor-Like Kinases, heat-shock and calcium-binding proteins to identify members of these gene families, whose basal expression under drought but potential SUMOylation presents them as QRF candidates for roles in stress signaling/response.