TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

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.

Top five unanswered questions in plant cell surface research.

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.

Genome wide association study of Arabidopsis seed mucilage layers at a regional scale.

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.

Evolutionary Analysis of Six Gene Families Part of the Reactive Oxygen Species (ROS) Gene Network in Three Brassicaceae Species.

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.

Cell-wall microdomain remodeling controls crucial developmental processes.

Plant cell walls display cellular and subcellular specificities. At the subcellular level, wall regional territories with specific compositions are necessary for macroscopic developmental processes. These regional specificities were named differently throughout the years, and are unified here under the term 'cell-wall microdomains' that define the local composition and organization of wall polymers underlying territories of wall loosening and/or softening or stiffening. We review 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.

Class III Peroxidases PRX01, PRX44, and PRX73 Control Root Hair Growth in Arabidopsis thaliana.

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.

Editorial for Special Issue: Research on Plant Cell Wall Biology.

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

Class III Peroxidases in Response to Multiple Abiotic Stresses in Arabidopsis thaliana Pyrenean Populations.

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.

Apoplastic class III peroxidases PRX62 and PRX69 promote Arabidopsis root hair growth at low temperature.

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.

Mixotrophy in aquatic plants, an overlooked ability.

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.

Effects of Dielectric Barrier Ambient Air Plasma on Two Brassicaceae Seeds: Arabidopsis thaliana and Camelina sativa.

We investigated low-temperature plasma effects on two Brassicaceae seeds (A. thaliana and C. sativa) using dielectric barrier discharge in air. Comparisons of plasma treatments on seeds showed distinct responses on germination rate and speed. Optimal treatment time giving optimal germination is 15 min for A. thaliana with 85% increase compared to control after 48 h of germination and 1 min for C. sativa with 75% increase compared to control after 32 h of germination. Such germination increases are associated with morphological changes shown by SEM of seed surface. For better understanding at the biochemical level, seed surfaces were analyzed using gas chromatography-mass spectrometry which underlined changes of lipidic composition. For both treated seeds, there is a decrease of saturated (palmitic and stearic) fatty acids while treated C. sativa showed a decrease of unsaturated (oleic and linoleic) acids and treated A. thaliana an increase of unsaturated ones. Such lipid changes, specifically a decrease of hydrophobic saturated fatty acids, are coherent with the other analyses (SEM, water uptake and contact angle). Moreover, an increase in A. thaliana of unsaturated acids (very reactive) probably neutralizes plasma RONS effects thus needing longer plasma exposure time (15 min) to reach optimal germination. For C. sativa, 1 min is enough because unsaturated linoleic acid becomes lower in treated C. sativa (1.2 × 107) compared to treated A. thaliana (3.7 × 107).

Myxospermy Evolution in Brassicaceae: A Highly Complex and Diverse Trait with Arabidopsis as an Uncommon Model.

The ability to extrude mucilage upon seed imbibition (myxospermy) occurs in several Angiosperm taxonomic groups, but its ancestral nature or evolutionary convergence origin remains misunderstood. We investigated seed mucilage evolution in the Brassicaceae family with comparison to the knowledge accumulated in Arabidopsis thaliana. The myxospermy occurrence was evaluated in 27 Brassicaceae species. Phenotyping included mucilage secretory cell morphology and topochemistry to highlight subtle myxospermy traits. In parallel, computational biology was driven on the one hundred genes constituting the so-called A. thaliana mucilage secretory cell toolbox to confront their sequence conservation to the observed phenotypes. Mucilage secretory cells show high morphology diversity; the three studied Arabidopsis species had a specific extrusion modality compared to the other studied Brassicaceae species. Orthologous genes from the A. thaliana mucilage secretory cell toolbox were mostly found in all studied species without correlation with the occurrence of myxospermy or even more sub-cellular traits. Seed mucilage may be an ancestral feature of the Brassicaceae family. It consists of highly diverse subtle traits, probably underlined by several genes not yet characterized in A. thaliana or by species-specific genes. Therefore, A. thaliana is probably not a sufficient reference for future myxospermy evo-devo studies.

Highlighting reactive oxygen species as multitaskers in root development.

Reactive oxygen species (ROS) are naturally produced by several redox reactions during plant regular metabolism such as photosynthesis and respiration. Due to their chemical properties and high reactivity, ROS were initially described as detrimental for cells during oxidative stress. However, they have been further recognized as key players in numerous developmental and physiological processes throughout the plant life cycle. Recent studies report the important role of ROS as growth regulators during plant root developmental processes such as in meristem maintenance, in root elongation, and in lateral root, root hair, endodermis, and vascular tissue differentiation. All involve multifaceted interplays between steady-state levels of ROS with transcriptional regulators, phytohormones, and nutrients. In this review, we attempt to summarize recent findings about how ROS are involved in multiple stages of plant root development during cell proliferation, elongation, and differentiation.

A powerful framework for an integrative study with heterogeneous omics data: from univariate statistics to multi-block analysis.

High-throughput data generated by new biotechnologies require specific and adapted statistical treatment in order to be efficiently used in biological studies. In this article, we propose a powerful framework to manage and analyse multi-omics heterogeneous data to carry out an integrative analysis. We have illustrated this using the mixOmics package for R software as it specifically addresses data integration issues. Our work also aims at applying the most recent functionalities of mixOmics to real datasets. Although multi-block integrative methodologies exist, we hope to encourage a more widespread use of such approaches in an operational framework by biologists. We have used natural populations of the model plant Arabidopsis thaliana in this work, but the framework proposed is not limited to this plant and can be deployed whatever the organisms of interest and the biological question may be. Four omics datasets (phenomics, metabolomics, cell wall proteomics and transcriptomics) were collected, analysed and integrated to study the cell wall plasticity of plants exposed to sub-optimal temperature growth conditions. The methodologies presented here start from basic univariate statistics leading to multi-block integration analysis. We have also highlighted the fact that each method, either unsupervised or supervised, is associated with one biological issue. Using this powerful framework enabled us to arrive at novel conclusions on the biological system, which would not have been possible using standard statistical approaches.

CsPrx25, a class III peroxidase in Citrus sinensis, confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification.

Citrus bacterial canker (CBC) results from Xanthomonas citri subsp. citri (Xcc) infection and poses a grave threat to citrus production. Class III peroxidases (CIII Prxs) are key proteins to the environmental adaptation of citrus plants to a range of exogenous pathogens, but the role of CIII Prxs during plant resistance to CBC is poorly defined. Herein, we explored the role of CsPrx25 and its contribution to plant defenses in molecular detail. Based on the expression analysis, CsPrx25 was identified as an apoplast-localized protein that is differentially regulated by Xcc infection, salicylic acid, and methyl jasmone acid in the CBC-susceptible variety Wanjincheng (C. sinensis) and the CBC-resistant variety Calamondin (C. madurensis). Transgenic Wanjincheng plants overexpressing CsPrx25 were generated, and these transgenic plants exhibited significantly increased CBC resistance compared with the WT plants. In addition, the CsPrx25-overexpressing plants displayed altered reactive oxygen species (ROS) homeostasis accompanied by enhanced H2O2 levels, which led to stronger hypersensitivity responses during Xcc infection. Moreover, the overexpression of CsPrx25 enhanced lignification as an apoplastic barrier for Xcc infection. Taken together, the results highlight how CsPrx25-mediated ROS homeostasis reconstruction and cell wall lignification can enhance the resistance of sweet orange to CBC.

The Class III Peroxidase Encoding Gene AtPrx62 Positively and Spatiotemporally Regulates the Low pH-Induced Cell Death in Arabidopsis thaliana Roots.

Exogenous low pH stress causes cell death in root cells, limiting root development, and agricultural production. Different lines of evidence suggested a relationship with cell wall (CW) remodeling players. We investigated whether class III peroxidase (CIII Prx) total activity, CIII Prx candidate gene expression, and reactive oxygen species (ROS) could modify CW structure during low pH-induced cell death in Arabidopsis thaliana roots. Wild-type roots displayed a good spatio-temporal correlation between the low pH-induced cell death and total CIII Prx activity in the early elongation (EZs), transition (TZs), and meristematic (MZs) zones. In situ mRNA hybridization showed that AtPrx62 transcripts accumulated only in roots treated at pH 4.6 in the same zones where cell death was induced. Furthermore, roots of the atprx62-1 knockout mutant showed decreased cell mortality under low pH compared to wild-type roots. Among the ROS, there was a drastic decrease in O2·- levels in the MZs of wild-type and atprx62-1 roots upon low pH stress. Together, our data demonstrate that AtPrx62 expression is induced by low pH and that the produced protein could positively regulate cell death. Whether the decrease in O2·- level is related to cell death induced upon low pH treatment remains to be elucidated.

An integrative Study Showing the Adaptation to Sub-Optimal Growth Conditions of Natural Populations of Arabidopsis thaliana: A Focus on Cell Wall Changes.

In the global warming context, plant adaptation occurs, but the underlying molecular mechanisms are poorly described. Studying natural variation of the model plant Arabidopsisthaliana adapted to various environments along an altitudinal gradient should contribute to the identification of new traits related to adaptation to contrasted growth conditions. The study was focused on the cell wall (CW) which plays major roles in the response to environmental changes. Rosettes and floral stems of four newly-described populations collected at different altitudinal levels in the Pyrenees Mountains were studied in laboratory conditions at two growth temperatures (22 vs. 15 °C) and compared to the well-described Col ecotype. Multi-omic analyses combining phenomics, metabolomics, CW proteomics, and transcriptomics were carried out to perform an integrative study to understand the mechanisms of plant adaptation to contrasted growth temperature. Different developmental responses of rosettes and floral stems were observed, especially at the CW level. In addition, specific population responses are shown in relation with their environment and their genetics. Candidate genes or proteins playing roles in the CW dynamics were identified and will deserve functional validation. Using a powerful framework of data integration has led to conclusions that could not have been reached using standard statistical approaches.

Coordination of five class III peroxidase-encoding genes for early germination events of Arabidopsis thaliana.

The Class III peroxidases (CIII Prxs) belong to a plant-specific multigene family. Thanks to their double catalytic cycle they can oxidize compounds or release reactive oxygen species (ROS). They are either involved in different cell wall stiffening processes such as lignification and suberization, in cell wall loosening or defense mechanisms. Germination is an important developmental stage requiring specific peroxidase activity. However, little is known about which isoforms are involved. Five CIII Prx encoding genes: AtPrx04, AtPrx16, AtPrx62, AtPrx69, and AtPrx71 were identified from published microarray data mining. Delayed or induced testa and endosperm rupture were observed for the corresponding CIII Prx mutant lines indicating either a gene-specific inducing or repressing role during germination, respectively. Via in situ hybridization AtPrx16, AtPrx62, AtPrx69 and AtPrx71 transcripts were exclusively localized to the micropylar endosperm facing the radicle, and transcriptomic data analysis enabled positioning the five CIII Prxs in a co-expression network enriched in germination, cell wall, cell wall proteins and xyloglucan hits. Evidence were produced showing that the five CIII Prxs were cell wall-targeted proteins and that the micropylar endosperm displayed a complex cell wall domain topochemistry. Finally, we drew a spatio-temporal model highlighting the fine sequential gene expression and the possible involvement of micropylar endosperm cell wall domains to explain the non-redundant cell wall stiffening and loosening functions of the CIII Prxs in a single cell type. We also highlighted the necessity of a peroxidase homeostasis to accurately control the micropylar endosperm cell wall dynamics during Arabidopsis germination events.