A developmentally controlled cellular decompartmentalization process executes programmed cell death in the Arabidopsis root cap.

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here, we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON (NAC) transcription factor SOMBRERO. Surprisingly, the breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.

Protoplast Preparation and Fluorescence-Activated Cell Sorting 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.

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.

Plant proteases during developmental programmed cell death.

Proteases are among the key regulators of most forms of programmed cell death (PCD) in animals. Many PCD processes have also been associated with protease expression or activation in plants, However, functional evidence for the roles and actual modes of action of plant proteases in PCD remains surprisingly limited. In this review, we provide 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 advances in the field, protease functions are often only correlatively linked to developmental PCD, and the specific molecular roles of proteases in many developmental PCD processes remain to be elucidated.

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.

ESCRT-mediated vesicle concatenation in plant endosomes.

Ubiquitinated plasma membrane proteins (cargo) are delivered to endosomes and sorted by endosomal sorting complex required for transport (ESCRT) machinery into endosome intralumenal vesicles (ILVs) for degradation. In contrast to the current model that postulates that ILVs form individually from inward budding of the endosomal limiting membrane, plant ILVs form as networks of concatenated vesicle buds by a novel vesiculation mechanism. We ran computational simulations based on experimentally derived diffusion coefficients of an ESCRT cargo protein and electron tomograms of Arabidopsis thaliana endosomes to measure cargo escape from budding ILVs. We found that 50% of the ESCRT cargo would escape from a single budding profile in 5-20 ms and from three concatenated ILVs in 80-200 ms. These short cargo escape times predict the need for strong diffusion barriers in ILVs. Consistent with a potential role as a diffusion barrier, we find that the ESCRT-III protein SNF7 remains associated with ILVs and is delivered to the vacuole for degradation.

Role of SKD1 Regulators LIP5 and IST1-LIKE1 in Endosomal Sorting and Plant Development.

SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRT components LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plants is not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasma membrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize the auxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cells over-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showed abnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts with IST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsis contains 12 genes coding for predicted IST1-domain containing proteins (ISTL1-12). Within the ISTL1-6 group, ISTL1 showed the strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through the analysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, is essential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality.

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

BACKGROUND AND AIMS: 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. METHODS: 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. KEY RESULTS: 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. CONCLUSIONS: 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.

Anatomy, ultrastructure and chemical composition of food bodies of Hovenia dulcis (Rhamnaceae).

BACKGROUND AND AIMS: Food bodies (FBs) are structures that promote mutualism between plants and ants, which help protect them against herbivores. The present study aims to describe the anatomical organization, ultrastructure and chemical composition of the FBs in Hovenia dulcis, which represent the first structures of this type described in Rhamnaceae. METHODS: Leaves in various stages of development were collected and fixed for examination under light, transmission and scanning electron microscopy. Samples of FBs were subjected to chemical analysis using thin-layer chromatography and nuclear magnetic resonance of (1)H and (13)C. KEY RESULTS: The FBs vary from globose to conical and are restricted to the abaxial leaf surface, having a mixed origin, including epidermis and parenchyma. The FB epidermis is uniseriate, slightly pilose and has a thin cuticle. The epidermal cells are vacuolated and pigments or food reserves are absent. The parenchyma cells of immature FBs have dense cytoplasm showing mitochondria, endoplasmic reticulum and plastids. Mature FB cells store oils, which are free in the cytosol and occupy a large portion of the cell lumen. In these cells the plastids accumulate starch. CONCLUSIONS: The lipids present in FBs are glycerin esters characteristic of plant energy reserves. Ants were observed collecting these FBs, which allows us to infer that these structures mediate plant-ant interactions and can help protect the young plants against herbivores, as these structures are prevalent at this developmental stage.