Abstract Many flowering plant species have adopted an aquatic lifestyle, contrasting with their terrestrial ancestors. Adapting to an aquatic environment required numerous evolutionary changes, including gene expansion and contraction. One of the most striking contractions has been observed in the genomes of seagrasses, where the ACO and ACS genes, involved in ethylene biosynthesis, are very few in number or even completely absent. To confirm this adaptation, we identified traces of gene loss in the genomes of four seagrass species, in the form of pseudogenes. Surprisingly, no gene loss was found in the species that had completely lost the function of ethylene synthesis, likely indicating an ancient loss of these genes. Conversely, several pseudogenes were found in the species where the ACO and ACS genes are contracting, indicating a recent and potentially ongoing process. We used the same approach on Utricularia gibba, a submerged freshwater plant, and also found a reduced number of ACO and ACS genes. In contrast, two terrestrial species closely related to seagrasses and U. gibba found a higher number of ACO and ACS genes, with no definitive evidence of gene loss. These results confirm that the loss of ethylene biosynthesis function in seagrasses is indeed linked to gene loss and suggests that it is an adaptation to a submerged rather than a marine lifestyle.

Terrestrial plants emerged from the water about 500 million years ago. Thereafter, they have diversified and now inhabit most of the Earth's surface. More recently, some species have re-adapted to an aquatic lifestyle, both in fresh and salt water, and fully or partially submerged. The mechanisms enabling these adaptations between terrestrial and aquatic life are extremely numerous, making it difficult to have a comprehensive overview of the phenomenon. Here, we performed a series of intraspecific measurements of the selection pressure affecting orthologous genes in eight aquatic and four terrestrial plants. Our analyses showed that aquatic plants have a relaxed selection pressure on nutrient assimilation mechanisms, probably linked to a greater bioavailability, as well as stronger adaptations to oxidative stress, while terrestrial plants evolution is linked to environment perception. Inter-species analyses have also highlighted a different evolution of chloroplast proteins between these two types of plants, suggesting adaptations to gas availability.

Abstract Floods cause significant crop losses worldwide. Plant response mechanisms to flooding have been extensively studied, particularly the ethylene-meditated mechanisms of perception and initiation of the response. However, other mechanisms are often studied more marginally, and it is difficult to determine which are species-specific and which are part of a conserved angiosperm response to hypoxia. Here, we performed a meta-analysis of transcriptomic data under hypoxic or flooding conditions across 11 angiosperm species and identified 259 homologous gene clusters that constitute the core response to hypoxia in angiosperms. These include the previously identified main mechanisms linked to ethylene, as well as numerous novel genes whose role in the hypoxia response is often poorly characterized. In particular, many previously overlooked genes associated with oxidative stress were identified as part of the core response, such as HRU1, TIP1-2, OZF1 , and OZF2 . Our results reveal many new candidate genes with strong potential for improving plant resilience to flooding.

Formerly considered as part of “junk DNA”, pseudogenes are nowadays known for their role in the post-transcriptional regulation of functional genes. Their identification also contributes to a better understanding of gene evolution, particularly in relation to adaptive responses and the evolution of multigene families. Despite this, there is, to our knowledge, no fully automatic pipeline allowing annotation of the pseudogenes on a whole genome. Here, we propose a new software named Pseudo-Gene Retriever (P-GRe). This is a completely automated pseudogene prediction tool requiring only a genome sequence, its corresponding GFF annotation file, and a protein sequences file. The aligner miniprot has been integrated in our pipeline, because of its high speed and sensitivity. With several filtering and post-analysis steps P-GRe outperforms existing software, while being more sensitive and bringing the new capacity of annotating unitary pseudogenes.

Heterophylly, characterized by distinct leaf morphologies, is an adaptive strategy evolved by some aquatic plants to cope with the contrasting environments in which they grow. It is shaped by environmental factors and by hormones, particularly abscisic and gibberellic acids. We conducted a comparative multi-omics analysis of submerged and floating leaves of Potamogeton nodosus, a heterophyllous species of the Alismatales order. Genome sequencing has confirmed its tetraploid status. A phylogenomic analysis has identified genes associated with aquatic adaptation, notably genes possibly involved in aurone biosynthesis, which are antioxidant flavonoids. Transcriptomic and metabolomic analyses have revealed distinct gene expression and metabolite patterns depending on the leaf position in the water column. The floating leaves exhibited upregulated genes associated with photosynthetic and metabolic activities, and with the accumulation of UV-protective metabolites reflecting light capture and photoprotection optimization. Conversely, submerged leaves showed upregulated genes associated with homeostatic processes, defense responses, and cell wall remodeling, reflecting tissue maintenance. We have also identified genes involved in the abscisic and gibberellic acids pathways. The developmental analyses highlighted active growth processes in young leaves. This multi-omic study provides an integrated understanding of the complex molecular mechanisms underlying the adaptation of P. nodosus to a dual lifestyle.

Abstract How the functional innovations that enabled plants to first colonize land 450 million years ago evolved remains puzzling. Here, we show that the gain of a single family of enzymes was pivotal in the evolution of two of these innovations, the cuticle that protect plants from dehydration and UV light, and the Arbuscular Mycorrhizal symbiosis promoting water and nutrient uptake. We show that functional phosphatase domain Glycerol-3-Phosphate Acyl Transferase (p-GPATs) evolved in the first land plants, concomitantly with the cuticle and symbiosis. Mutation of two p-GPATs from the liverwort Marchantia paleacea is sufficient to abolish both cuticle formation and AM symbiosis, leading to major developmental defects. We propose that the evolution of p-GPATs in land plants acted as a two birds-one stone innovation, diverting intracellular lipids to the extracellular space and providing a simple path to the evolution of two traits essential for the colonization of land.

Obtaining in vitro plant cultures free of parasites or pathogens, namely axenic cultures is a major challenge both in the field of scientific research and in agronomy or biotechnology. For aquatic macrophytes, plants free from contamination are required for aquaculture or aquariums, and necessary to prevent microbial growth in medium containing dissolved organic carbon. Then, various protocols have been elaborated to facilitate the production of different qualities of plant cultures from "clean" to sterile. The performance of various calcium hypochlorite axenisation protocols in aquatic plants was evaluated. Through trials with variations in hypochlorite concentration and exposure time, it became possible to draw up a response table for three first species (Ceratophyllum demersum, Lemna minor and Myriophyllum spicatum). Additional results on other species were used to define the response of different species to axenisation, so that each species could be oriented towards a particular technique. A trial with successive axenisation steps was thus successfully tested on the two branchy but resilient species (C. demersum and M. spicatum). The effectiveness of protocol variations (e.g. addition of sucrose and use of rinsing breaks during hypochlorite exposures) were also evaluated. This resulted in recommendations that we propose, primarily the banning of sugars during the early stages of axenisation, contrary to previous recommendations in literature protocols.

Plant adaptation to terrestrial life started 450 million years ago and has played a major role in the evolution of life on Earth. The genetic mechanisms allowing this adaptation to a diversity of terrestrial constraints have been mostly studied by focusing on flowering plants. Here, we gathered a collection of 133 accessions of the model bryophyte Marchantia polymorpha and studied its intraspecific diversity using selection signature analyses, a genome-environment association study and a pangenome. We identified adaptive features, such as peroxidases or nucleotide-binding and leucine-rich repeats (NLRs), also observed in flowering plants, likely inherited from the first land plants. The M. polymorpha pangenome also harbors lineage-specific accessory genes absent from seed plants. We conclude that different land plant lineages still share many elements from the genetic toolkit evolved by their most recent common ancestor to adapt to the terrestrial habitat, refined by lineage-specific polymorphisms and gene family evolution.

The myxospermous species Arabidopsis thaliana extrudes a polysaccharidic mucilage from the seed coat epidermis during imbibition. The whole seed mucilage can be divided into a seed-adherent layer and a fully soluble layer, both layers presenting natural genetic variations. The adherent mucilage is variable in size and composition, while the soluble mucilage is variable in composition and physical properties. Studies reporting both the genetic architecture and the putative selective agents acting on this natural genetic variation are scarce. In this study, we set up a Genome Wide Association study (GWAS) based on 424 natural accessions collected from 166 natural populations of A. thaliana located south-west of France and previously characterized for a very important number of abiotic and biotic factors. We identified an extensive genetic variation for both mucilage layers. The adherent mucilage was mainly related to precipitation and temperature whereas the non-adherent mucilage was unrelated to any environmental factors. By combining a hierarchical Bayesian model with a local score approach, we identified 55 and 28 candidate genes, corresponding to 26 and 10 QTLs for the adherent and non-adherent mucilages, respectively. Putative or characterized function and expression data available in the literature were used to filter the candidate genes. Only one gene among our set of candidate genes was already described as a seed mucilage actor, leaving a large set of new candidates putatively implicated inseed mucilage synthesis or release. The present study lay out foundation to understand the influence of regional ecological factors acting on seed mucilage in A. thaliana.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Climate change is expected to intensify the occurrence of abiotic stress in plants, such as hypoxia and salt stresses, leading to the production of reactive oxygen species (ROS), which need to be effectively managed by various oxido-reductases encoded by the so-called ROS gene network. Here, we studied six oxido-reductases families in three Brassicaceae species, Arabidopsis thaliana as well as Nasturtium officinale and Eutrema salsugineum, which are adapted to hypoxia and salt stress, respectively. Using available and new genomic data, we performed a phylogenomic analysis and compared RNA-seq data to study genomic and transcriptomic adaptations. This comprehensive approach allowed for the gaining of insights into the impact of the adaptation to saline or hypoxia conditions on genome organization (gene gains and losses) and transcriptional regulation. Notably, the comparison of the N. officinale and E. salsugineum genomes to that of A. thaliana highlighted changes in the distribution of ohnologs and homologs, particularly affecting class III peroxidase genes (CIII Prxs). These changes were specific to each gene, to gene families subjected to duplication events and to each species, suggesting distinct evolutionary responses. The analysis of transcriptomic data has allowed for the identification of genes related to stress responses in A. thaliana, and, conversely, to adaptation in N. officinale and E. salsugineum.

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

Formerly considered as part of “junk DNA”, pseudogenes are nowadays known for their role in the post-transcriptional regulation of functional genes. In addition, their identification allows a better understanding of gene evolution in the frame of multigenic families. Despite this, there is, to our knowledge, no fully automatic user-friendly software allowing the annotation of pseudogenes on a whole genome. Here, we present Pseudo-Gene Retriever (P-GRe), a fully automated pseudogene prediction software requiring only a genome sequence and its corresponding GFF annotation file. P-GRe detects the sequences of the pseudogenes on a whole genome and returns to the user all their genomic sequences and their pseudo-coding sequences. The ability of P-GRe to finely reconstruct the structure of pseudogenes also allow to obtain a set of proteins virtually encoded by the predicted pseudogenes. We show here that in 70% of the cases, virtual proteins constructed by P-GRe from Arabidopsis thaliana proteome and genome aligned better to their parent protein than their annotated counterpart. ### Competing Interest Statement The authors have declared no competing interest.

Plant adaptation to a terrestrial life 450 million years ago played a major role in the evolution of life on Earth. This shift from an aquatic environment has been mostly studied by focusing on flowering plants. Here, we gathered a collection of 133 accessions of the non-vascular plants Marchantia polymorpha and studied its intraspecific diversity using selection signature analyses, genome-environment association study and a gene-centered pangenome. We identified adaptive features shared with flowering plants, such as peroxidases or nucleotide-binding and leucine-rich repeat (NLR), which likely played a role in the adaptation of the first land plants to the terrestrial habitat. The M. polymorpha pangenome also harbored lineage-specific accessory genes absent from seed plants. We conclude that different land plants lineages still share many elements from the genetic toolkit evolved by their most recent common ancestor to adapt to the terrestrial habitat, refined by lineage specific polymorphisms and gene family evolutions.

Root Hairs (RHs) growth is influenced by endogenous and by external environmental signals that coordinately regulate its final cell size. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explore the molecular basis of this RH growth response by using a Genome Wide Association Study (GWAS) approach using Arabidopsis thaliana natural accessions. We identify the poorly characterized PEROXIDASE 62 (PRX62) and a related protein PRX69 as key proteins under moderate low temperature stress. Strikingly, a cell wall protein extensin (EXT) reporter reveals the effect of peroxidase activity on EXT cell wall association at 10 °C in the RH apical zone. Collectively, our results indicate that PRX62, and to a lesser extent PRX69, are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low temperature.

Plant cells are surrounded by extracellular matrixes [...].

Aquatic Embryophytes play a key role in the proper functioning of aquatic ecosystems, where carbon (inorganic and organic forms) is pivotal in biogeochemical processes. There is growing awareness that mixotrophy, the direct use of exogenous organic carbon by autotrophs, is a widespread phenomenon and that it has emerged recurrently in the evolution of many autotrophic lineages. Despite living in an environment providing organic matter and presenting many favourable predispositions, aquatic plants from the Embryophytes, except carnivorous ones, have never been deeply investigated for mixotrophy. Here, we address the possibility that aquatic plants may exhibit mixotrophy, a prospect overlooked by research until now, and that this may be much more widespread than imagined under the conventional paradigm of plants considered as strict autotrophs.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.

Class III peroxidases constitute a plant-specific multigene family, where 73 genes have been identified in Arabidopsis thaliana. These genes are members of the reactive oxygen species (ROS) regulatory network in the whole plant, but more importantly, at the root level. In response to abiotic stresses such as cold, heat, and salinity, their expression is significantly modified. To learn more about their transcriptional regulation, an integrative phenotypic, genomic, and transcriptomic study was executed on the roots of A. thaliana Pyrenean populations. Initially, the root phenotyping highlighted 3 Pyrenean populations to be tolerant to cold (Eaux), heat (Herr), and salt (Grip) stresses. Then, the RNA-seq analyses on these three populations, in addition to Col-0, displayed variations in CIII Prxs expression under stressful treatments and between different genotypes. Consequently, several CIII Prxs were particularly upregulated in the tolerant populations, suggesting novel and specific roles of these genes in plant tolerance against abiotic stresses.

Plant cell walls display cellular and subcellular specificities. At the subcellular level, wall regional territories with specific compositions are necessary for mac-roscopic developmental processes. These regional specificities were named dif-ferently throughout the years, and are unified here under the term 'cell-wall microdomains' that define the local composition and organization of wall poly-mers underlying territories of wall loosening and/or softening or stiffening. We re-view the occurrence and developmental role of wall microdomains in different cell types. We primarily focus on the contribution of two categories of wall -remodeling molecular actors: fine-tuning of homogalacturonan (HG; pectin) demethylesterification patterns and two classes of oxidoreductases [class III peroxidases (CIII PRXs) and laccases (LACs)], but we also highlight two different molecular scaffolds recently identified for positioning specific CIII PRXs.