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.

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.

Seed coats as an alternative molecular factory: thinking outside the box.

KEY MESSAGE: Seed coats as commodities. Seed coats play important roles in the protection of the embryo from biological attack and physical damage by the environment as well as dispersion strategies. A significant part of the energy devoted by the mother plant to seed production is channeled into the production of the cell layers and metabolites that surround the embryo. Nevertheless, in crop species these are often discarded post-harvest and are a wasted resource that could be processed to yield co-products. The production of novel compounds from existing metabolites is also a possibility. A number of macromolecules are already accumulated in these maternal layers that could be exploited in industrial applications either directly or via green chemistry, notably flavonoids, lignin, lignan, polysaccharides, lipid polyesters and waxes. Here, we summarize our knowledge of the in planta biosynthesis pathways of these macromolecules and their molecular regulation as well as potential applications. We also outline recent work aimed at providing further tools for increasing yields of existing molecules or the development of novel biotech approaches, as well as trial studies aimed at exploiting this underused resource.

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.

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.

Roles of cell wall peroxidases in plant development.

Class III peroxidases (CIII Prxs) are plant specific proteins. Based on in silico prediction and experimental evidence, they are mainly considered as cell wall localized proteins. Thanks to their dual hydroxylic and peroxidative cycles, they can produce ROS as well as oxidize cell wall aromatic compounds within proteins and phenolics that are either free or linked to polysaccharides. Thus, they are tightly associated to cell wall loosening and stiffening. They are members of large multigenic families, mostly due to an elevated rate of gene duplication in higher plants, resulting in a high risk of functional redundancy between them. However, proteomic and (micro)transcriptomic analyses have shown that CIII Prx expression profiles are highly specific. Based on these omic analyses, several reverse genetic studies have demonstrated the importance of the spatio-temporal regulation of their expression and ability to interact with cell wall microdomains in order to achieve specific activity in vivo. Each CIII Prx isoform could have specific functions in muro and this could explain the conservation of a high number of genes in plant genomes.

Expression of PRX36, PMEI6 and SBT1.7 is controlled by complex transcription factor regulatory networks for proper seed coat mucilage extrusion.

Mucilage secretory cells (MSC) form an intriguing cell layer important for seed germination. In Arabidopsis thaliana, several master transcription factors (TFs) and "actor" proteins have already been identified as key players for seed coat differentiation including epidermal cell formation, mucilage production and extrusion. The regulation of the genes coding for MSC cell wall "actor" proteins by TFs needs to be better established. Here, the expression and the regulation of 3 known actors (PRX36, PMEI6, SBT1.7) and 2 additional putative actors (PRX56, DIR12) have been analyzed in T-DNA mutants affected in master TFs (ap2, egl3/gl3, gl2, myb5, tt8, ttg1, ttg2 and luh1/mum1). Genes with somehow similar function are differentially regulated and conversely, genes with different functions are regulated in similar manner.