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.

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.

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.

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.

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.

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.

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.

Global analysis of non-animal peroxidases provides insights into the evolution of this gene family in the green lineage.

The non-animal peroxidases belong to a superfamily of oxidoreductases that reduce hydrogen peroxide and oxidize numerous substrates. Since their initial characterization in 1992, a number of studies have provided an understanding of the origin and evolution of this protein family. Here, we report a comprehensive evolutionary analysis of non-animal peroxidases using integrated in silico and biochemical approaches. Thanks to the availability of numerous genomic sequences from more than 2500 species belonging to 14 kingdoms together with expert and comprehensive annotation of peroxidase sequences that have been centralized in a dedicated database, we have been able to use phylogenetic reconstructions to increase our understanding of the evolutionary processes underlying the diversification of non-animal peroxidases. We analysed the distribution of all non-animal peroxidases in more than 200 eukaryotic organisms in silico. First, we show that the presence or absence of non-animal peroxidases correlates with the presence or absence of certain organelles or with specific biological processes. Examination of almost 2000 organisms determined that ascorbate peroxidases (APxs) and cytochrome c peroxidases (CcPs) are present in those containing chloroplasts and mitochondria, respectively. Plants, which contain both organelles, are an exception and contain only APxs without CcP. Class II peroxidases (CII Prxs) are only found in fungi with wood-decay and plant-degradation abilities. Class III peroxidases (CIII Prxs) are only found in streptophyte algae and land plants, and have been subjected to large family expansion. Biochemical activities of APx, CcP, and CIII Prx assessed using protein extracts from 30 different eukaryotic organisms support the distribution of the sequences resulting from our in silico analysis. The biochemical results confirmed both the presence and classification of the non-animal peroxidase encoding sequences.

Transcriptomic and cell wall proteomic datasets of rosettes and floral stems from five Arabidopsis thaliana ecotypes grown at optimal or sub-optimal temperature.

This article provides experimental data describing the RNA and the cell wall protein profiles of rosettes and flower stems of five Arabidopsis thaliana ecotypes. Four newly-described Pyrenees ecotypes [1] are analyzed in addition to the well-described and sequenced Columbia (Col) ecotype of A. thaliana. All five ecotypes have been grown at two different temperatures, 22 °C and 15 °C. We provide transcriptomics and cell wall proteomics data regarding (i) rosettes at the bolting stage, and (ii) floral stems at the first flower stage. These data are a valuable resource to study the adaptation of A. thaliana ecotypes to sub-optimal temperature growth conditions.

Medium-Throughput RNA In Situ Hybridization of Serial Sections from Paraffin-Embedded Tissue Microarrays.

(m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This "one tissue-numerous genes" information can be completed by the reverse "one gene-numerous tissues" picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-throughput ISH protocol performed on serial sections from tissue microarrays. In a recent work, we implemented this method for 39 selected genes studied by medium-throughput ISH complementing an existing tissue-specific transcriptomic dataset focused on the model plant Arabidopsis seed development kinetics (Francoz et al., Scientific Reports 6:24644, 2016). This full integration of ISH and transcriptomics demonstrated the complementarity of both techniques in terms of tissue/cell specificity, signal sensitivity, gene specificity, and spatiotemporal resolution.

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.

Pectin Demethylesterification Generates Platforms that Anchor Peroxidases to Remodel Plant Cell Wall Domains.

Plant cell walls are made of polysaccharidic-proteinaceous complex matrices. Molecular interactions governing their organization remain understudied. We take advantage of the highly dynamic cell walls of Arabidopsis seed mucilage secretory cells to propose a hierarchical multi-molecular interaction model within a cell wall domain. We show that the PECTINMETHYLESTERASE INHIBITOR6 activity creates a partially demethylesterified pectin pattern acting as a platform allowing positioning of PEROXIDASE36 in a remote primary cell wall domain during early development. This allows triggering the loosening of this domain during later development, in turn leading to proper physiological function upon mature seed imbibition and germination. We anticipate that this pioneer example of molecular scaffold within a cell wall domain is more widespread through other combinations of the individual molecular players all belonging to large multigenic families. These results highlight the role of cell wall polysaccharide-protein interactions in the organization of cell wall domains.

Cell wall modifications of two Arabidopsis thaliana ecotypes, Col and Sha, in response to sub-optimal growth conditions: An integrative study.

With the global temperature change, plant adaptations are predicted, but little is known about the molecular mechanisms underlying them. Arabidopsis thaliana is a model plant adapted to various environmental conditions, in particular able to develop along an altitudinal gradient. Two ecotypes, Columbia (Col) growing at low altitude, and Shahdara (Sha) growing at 3400m, have been studied at optimal and sub-optimal growth temperature (22°C vs 15°C). Macro- and micro-phenotyping, cell wall monosaccharides analyses, cell wall proteomics, and transcriptomics have been performed in order to accomplish an integrative analysis. The analysis has been focused on cell walls (CWs) which are assumed to play roles in response to environmental changes. At 15°C, both ecotypes presented characteristic morphological traits of low temperature growth acclimation such as reduced rosette diameter, increased number of leaves, modifications of their CW composition and cuticle reinforcement. Altogether, the integrative analysis has allowed identifying several candidate genes/proteins possibly involved in the cell wall modifications observed during the temperature acclimation response.

Molecular link between auxin and ROS-mediated polar growth.

Root hair polar growth is endogenously controlled by auxin and sustained by oscillating levels of reactive oxygen species (ROS). These cells extend several hundred-fold their original size toward signals important for plant survival. Although their final cell size is of fundamental importance, the molecular mechanisms that control it remain largely unknown. Here we show that ROS production is controlled by the transcription factor RSL4, which in turn is transcriptionally regulated by auxin through several auxin response factors (ARFs). In this manner, auxin controls ROS-mediated polar growth by activating RSL4, which then up-regulates the expression of genes encoding NADPH oxidases (also known as RESPIRATORY BURST OXIDASE HOMOLOG proteins) and class III peroxidases, which catalyze ROS production. Chemical or genetic interference with ROS balance or peroxidase activity affects root hair final cell size. Overall, our findings establish a molecular link between auxin and ROS-mediated polar root hair growth.

The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation.

Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using ß-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.

Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics.

The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

High-throughput microanalysis of large lignocellulosic sample sets by pyrolysis-gas chromatography/mass spectrometry.

High-throughput analytical techniques to assess the chemistry of lignocellulosic plant material are crucial to plant cell-wall research. We have established an analytical platform for this purpose and demonstrated its usefulness with two applications. The system is based on analytical pyrolysis, coupled to gas chromatography/mass spectrometry - a technique particularly suited for analysis of lignocellulose. Automated multivariate-based data-processing methods are used to obtain results within a few hours after analysis, with an experimental batch of 500 analyzed samples. The usefulness of multivariate sample discrimination methods and hierarchical clustering of samples is demonstrated. We have analyzed an Arabidopsis mutant collection consisting of 300 samples representing 31 genotypes. The mutant collection is presented through cluster analysis, based on chemotypic difference, with respect to wild type. Further, we have analyzed 500 thin sections from five biological replicate trees to create a spatial highly resolved profile of the proportions of syringyl-, guaiacyl- and p-hydroxyphenyl lignin across phloem, developing and mature wood in aspen. The combination of biologically easy to interpret information, the low demand of sample amount and the flexibility in sample types amenable to analysis makes this technique a valuable extension to the range of established high-throughput biomaterial analytical platforms.

The Arabidopsis Class III Peroxidase AtPRX71 Negatively Regulates Growth under Physiological Conditions and in Response to Cell Wall Damage.

The structure of the cell wall has a major impact on plant growth and development, and alteration of cell wall structural components is often detrimental to biomass production. However, the molecular mechanisms responsible for these negative effects are largely unknown. Arabidopsis (Arabidopsis thaliana) plants with altered pectin composition because of either the expression of the Aspergillus niger polygalacturonase II (AnPGII; 35S:AnPGII plants) or a mutation in the QUASIMODO2 (QUA2) gene that encodes a putative pectin methyltransferase (qua2-1 plants), display severe growth defects. Here, we show that expression of Arabidopsis PEROXIDASE71 (AtPRX71), encoding a class III peroxidase, strongly increases in 35S:AnPGII and qua2-1 plants as well as in response to treatments with the cellulose synthase inhibitor isoxaben, which also impairs cell wall integrity. Analysis of atprx71 loss-of-function mutants and plants overexpressing AtPRX71 indicates that this gene negatively influences Arabidopsis growth at different stages of development, likely limiting cell expansion. The atprx71-1 mutation partially suppresses the dwarf phenotype of qua2-1, suggesting that AtPRX71 contributes to the growth defects observed in plants undergoing cell wall damage. Furthermore, AtPRX71 seems to promote the production of reactive oxygen species in qua2-1 plants as well as plants treated with isoxaben. We propose that AtPRX71 contributes to strengthen cell walls, therefore restricting cell expansion, during normal growth and in response to cell wall damage.

Arabidopsis seed mucilage secretory cells: regulation and dynamics.

Seeds from various angiosperm species produce polysaccharide mucilage facilitating germination and, therefore, conferring major evolutionary advantages. The seed epidermal mucilage secretory cells (MSCs) undergo numerous tightly controlled changes of their extracellular matrixes (ECMs) throughout seed development. Recently, major progress based on the model species Arabidopsis thaliana was published, including the identification of 54 genes necessary for mucilage synthesis and release. Here, we review these genes that constitute the so-called 'MSC toolbox', within which transcription factors and proteins related to polysaccharide production, secretion, modification, and stabilization are the most abundant and belong to complex regulatory networks. We also discuss how seed coat 'omics data-mining, comparative genomics, and operon-like gene cluster studies will provide means to identify new members of the MSC toolbox.