Plant proteases during developmental programmed cell death

Proteases are among the key regulators of most forms of programmed cell death (PCD) in animals. Also in plants, many PCD processes have been associated with protease expression or activation. However, the functional evidence of the roles and actual modes of action of plant proteases in PCD remains surprisingly limited. In this review, we give an update on protease involvement in the context of developmentally regulated plant PCD. To illustrate the diversity of protease functions, we focus on several prominent developmental PCD processes, including xylem and tapetum maturation, suspensor elimination, endosperm degradation and seed coat formation, as well as plant senescence processes. Despite the substantial advance in the field, protease functions are still often only correlatively linked to developmental PCD, and the specific molecular roles of proteases in many developmental PCD processes remain to be elucidated

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

CRISPR technology is an established tool for the generation of knockout plants (Zhang et al., 2019), yet limitations remain. First, the manipulation of individual genes may fail to produce phenotypes for groups of genes with redundant or synergistic functions. While this has been partially addressed by multiplexing guide RNAs (gRNAs), there is concern that as the number of targets increases, the chances of obtaining higher-order knockouts diminish (Zhang et al., 2016). Second, knocking out fundamentally important genes can cause severe pleiotropic phenotypes or lethality. Tissue-specific knockout of genes in somatic tissues can overcome this limitation (Decaestecker et al., 2019 ; Wang et al., 2020 ; Liang et al., 2019). However, the efficiency of simultaneously targeting more than three genes in a tissue-specific context is unexplored. Here, by multiplexing gRNAs in Arabidopsis thaliana plants expressing Cas9 either ubiquitously (pPcUBI) or root cap-specifically (pSMB), we show that six genes can be simultaneously mutated with high efficiency, generating higher-order mutant phenotypes already in the first transgenic generation (T1). The mutation frequencies for all target genes were positively correlated and unaffected by the order of the gRNAs in the vector, showing that efficient higher-order mutagenesis in specific plant tissues can be readily achieved

CRISPR-TSKO : a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function

NAC transcription factors ANAC087 and ANAC046 control distinct aspects of programmed cell death in the Arabidopsis columella and lateral root cap

Programmed cell death in plants occurs both during stress responses and as an integral part of regular plant development. Despite the undisputed importance of developmentally controlled cell death processes for plant growth and reproduction, we are only beginning to understand the underlying molecular genetic regulation. Exploiting the Arabidopsis thaliana root cap as a cell death model system, we identified two NAC transcription factors, the little-characterized ANAC087 and the leaf-senescence regulator ANAC046, as being sufficient to activate the expression of cell death-associated genes and to induce ectopic programmed cell death. In the root cap, these transcription factors are involved in the regulation of distinct aspects of programmed cell death. ANAC087 orchestrates postmortem chromatin degradation in the lateral root cap via the nuclease BFN1. In addition, both ANAC087 and ANAC046 redundantly control the onset of cell death execution in the columella root cap during and after its shedding from the root tip. Besides identifying two regulators of developmental programmed cell death, our analyses reveal the existence of an actively controlled cell death program in Arabidopsis columella root cap cells

WorkflowHub - A FAIR registry for workflows

<p>Poster on WorkflowHub presented at the ELIXIR Belgium Conference, 2023: "ELIXIR Belgium: Your data, Our services, European success".</p&gt

Food bodies in Cissus verticillata (Vitaceae): ontogenesis, structure and functional aspects

The distinction between pearl bodies (or pearl glands) and food bodies (FBs) is not clear; neither is our understanding of what these structures really represent. The present work examined the ontogenesis, structure, ultrastructure and histochemical aspects of the protuberances in Cissus verticillata, which have been described since the beginning of the 19th century as pearl glands or pearl bodies, in order to establish a relationship between their structure and function.Segments of stems and leaves in different stages of development were collected and fixed for study under light microscopy as well as electron transmission and scanning microscopy. Samples of FBs were subjected to chemical analysis using thin-layer chromatography.The FBs in C. verticillata are globose and attached to the plant by a short peduncle. These structures are present along the entire stem during primary growth, and on the inflorescence axis and the abaxial face of the leaves. The FBs were observed to be of mixed origin, with the participation of both the epidermis and the underlying parenchymatic cells. The epidermis is uniseriate with a thin cuticle, and the cells have dense cytoplasm and a large nucleus. The internal parenchymatic cells have thin walls; in the young structures these cells have dense cytoplasm with a predominance of mitochondria and plastids. In the mature FBs, the parenchymatic cells accumulate oils and soluble sugars; dictyosomes and rough endoplasmic reticulum predominate in the cytoplasm; the vacuoles are ample. Removal of the FBs appears to stimulate the formation of new ones, at the same place.The vegetative vigour of the plant seems to influence the number of FBs produced, with more vigorous branches having greater densities of FBs. The results allow the conclusion that the structures traditionally designated pearl glands or pearl bodies in C. verticillata constitute FBs that can recruit large numbers of ants.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Protoplast preparation and fluorescence-activated cell for the evaluation of targeted mutagenesis in plant cells

Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells

Umbrella Data Management Plans to integrate FAIR data:Lessons from the ISIDORe and BY-COVID consortia for pandemic preparedness

The Horizon Europe project ISIDORe is dedicated to pandemic preparedness and responsiveness research. It brings together 17 Research Infrastructures (RIs) and networks to provide a broad range of services to infectious disease researchers. An efficient and structured treatment of data is central to ISIDORe’s aim to furnish seamless access to its multidisciplinary catalogue of services, and to ensure that users’ results are treated FAIRly. ISIDORe therefore requires a data management plan (DMP) covering both access management and research outputs, applicable over a broad range of disciplines, and compatible with the constraints and existing practices of its diverse partners. We undertook an iterative, step-by-step, process to build a community-approved living document, identifying good practices and processes, on the basis of use cases, presented as proof of concepts. International fora such as the RDA and EOSC, and primarily the BY-COVID project, furnished registries, tools and online data platforms, as well as standards, and the support of data scientists. Together, these elements provide a path for building an umbrella, FAIR-compliant DMP, aligned as fully as possible with FAIR principles, which could also be applied as a framework for data management harmonisation in other large-scale, challenge-driven projects. Finally, we discuss how data management and reuse can be further improved through the writing of realistic DMPs using 'DMP profiles' and, in the future, the contribution of an inter RIs data steward network, to produce a Community of Practice that could be integrated into planned trans-RI competence centres.Preprint added on zenodo with the autorisation of DSJ Edito