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.

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

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.

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.

Complex regulation of prolyl-4-hydroxylases impacts root hair expansion.

Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycoproteins, including extensins (EXTs). Proline hydroxylation, an early post-translational modification (PTM) of HRGPs catalyzed by prolyl 4-hydroxylases (P4Hs), defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

ER network homeostasis is critical for plant endosome streaming and endocytosis.

Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER-endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER-endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network.

A novel endosomal sorting complex required for transport (ESCRT) component in Arabidopsis thaliana controls cell expansion and development.

ESCRT proteins mediate membrane remodeling and scission events and are essential for endosomal sorting of plasma membrane proteins for degradation. We have identified a novel, plant-specific ESCRT component called PROS (POSITIVE REGULATOR OF SKD1) in Arabidopsis thaliana. PROS has a strong positive effect on the in vitro ATPase activity of SKD1 (also known as Vacuolar Protein Sorting 4 or VPS4), a critical component required for ESCRT-III disassembly and endosomal vesiculation. PROS interacts with both SKD1 and the SKD1-positive regulator LIP5/VTA1. We have identified a putative MIM domain within PROS that mediate the interaction with the MIT domain of SKD1. Interestingly, whereas MIM domains are commonly found at the C terminus of ESCRT-III subunits, the PROS MIM domain is internal. The heterologous expression of PROS in yeast mutant cells lacking Vta1p partially rescues endosomal sorting defects. PROS is expressed in most tissues and cells types in Arabidopsis thaliana. Silencing of PROS leads to reduced cell expansion and abnormal organ growth.

Plant endosomal trafficking pathways.

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.

The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development.

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

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.