ABSTRACT
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.
Subject(s)
Arabidopsis/growth & development , Plant Root Cap/metabolism , Plant Root Cap/physiology , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Cell Wall/metabolism , Gene Expression Regulation, Plant/genetics , Membrane Lipids/biosynthesis , Membrane Lipids/metabolism , Meristem/metabolism , Mutation , Plant Roots/cytology , Seedlings/genetics , Seedlings/growth & developmentABSTRACT
Programmed cell death (PCD) is a collective term for diverse processes causing an actively induced, tightly controlled cellular suicide. PCD has a multitude of functions in the development and health of multicellular organisms. In comparison to intensively studied forms of animal PCD such as apoptosis, our knowledge of the regulation of PCD in plants remains limited. Despite the importance of PCD in plant development and as a response to biotic and abiotic stresses, the complex molecular networks controlling different forms of plant PCD are only just beginning to emerge. With this review, we provide an update on the considerable progress that has been made over the last decade in our understanding of PCD as an inherent part of plant development. We highlight both functions of developmental PCD and central aspects of its molecular regulation.
Subject(s)
Apoptosis , Plant Development , Cellular Senescence , Plant Cells/metabolism , ReproductionABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Calcium/metabolism , Apoptosis/physiology , Cell DeathABSTRACT
Flowers have a species-specific fertile period during which pollination and fertilization have to occur to initiate seed and fruit development. Unpollinated flowers remain receptive for mere hours in some species, and up to several weeks in others before flower senescence terminates fertility. As such, floral longevity is a key trait subject to both natural selection and plant breeding. Within the flower, the life span of the ovule containing the female gametophyte is decisive for fertilization and the initiation of seed development. Here, we show that unfertilized ovules in Arabidopsis thaliana undergo a senescence program that generates morphological and molecular hallmarks of canonical programmed cell death processes in the sporophytically derived ovule integuments. Transcriptome profiling of isolated aging ovules revealed substantial transcriptomic reprogramming during ovule senescence, and identified up-regulated transcription factors as candidate regulators of these processes. Combined mutation of three most-up-regulated NAC (NAM, ATAF1/2, and CUC2) transcription factors, NAP/ANAC029, SHYG/ANAC047, and ORE1/ANAC092, caused a substantial delay in ovule senescence and an extension of fertility in Arabidopsis ovules. These results suggest that timing of ovule senescence and duration of gametophyte receptivity are subject to genetic regulation controlled by the maternal sporophyte.
Subject(s)
Arabidopsis , Transcription Factors , Ovule , Plant Breeding , FertilityABSTRACT
Plant flowers have a functional life span during which pollination and fertilization occur to ensure seed and fruit development. Once flower senescence is initiated, the potential to set seed or fruit is irrevocably lost. In maize, silk strands are the elongated floral stigmas that emerge from the husk-enveloped inflorescence to intercept airborne pollen. Here we show that KIRA1-LIKE1 (KIL1), an ortholog of the Arabidopsis NAC (NAM (NO APICAL MERISTEM), ATAF1/2 (Arabidopsis thaliana Activation Factor1 and 2) and CUC (CUP-SHAPED COTYLEDON 2)) transcription factor KIRA1, promotes senescence and programmed cell death (PCD) in the silk strand base, ending the window of accessibility for fertilization of the ovary. Loss of KIL1 function extends silk receptivity and thus strongly increases kernel yield following late pollination. This phenotype offers new opportunities for possibly improving yield stability in cereal crops. Moreover, despite diverging flower morphologies and the substantial evolutionary distance between Arabidopsis and maize, our data indicate remarkably similar principles in terminating floral receptivity by PCD, whose modulation offers the potential to be widely used in agriculture.
Subject(s)
Arabidopsis , Arabidopsis/physiology , Fertility/genetics , Flowers/physiology , Gene Expression Regulation, Plant/genetics , Silk/genetics , Silk/metabolism , Zea mays/genetics , Zea mays/metabolismABSTRACT
Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Endocytosis , Gene Expression Regulation, Plant , Meristem/genetics , Plants/metabolism , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells.
Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , DNA Methylation/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , Single-Cell Analysis , Animals , Arabidopsis Proteins/metabolism , Cell Line , DNA-Directed RNA Polymerases/metabolism , Embryonic Stem Cells , Gene Expression Regulation , Mice , Plants, Genetically Modified , Reproduction/genetics , Sex FactorsABSTRACT
Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.
Subject(s)
Apoptosis , Research , Plants , Plant Cells/physiologyABSTRACT
Developmental programmed cell death (dPCD) controls a plethora of functions in plant growth and reproduction. In the root cap of Arabidopsis (Arabidopsis thaliana), dPCD functions to control organ size in balance with the continuous stem cell activity in the root meristem. Key regulators of root cap dPCD including SOMBRERO/ANAC033 (SMB) belong to the NAC family of transcription factors. Here, we identify the C2H2 zinc finger protein ZINC FINGER OF ARABIDOPSIS THALIANA 14 ZAT14 as part of the gene regulatory network of root cap dPCD acting downstream of SMB. Similar to SMB, ZAT14-inducible misexpression leads to extensive ectopic cell death. Both the canonical EAR motif and a conserved L-box motif of ZAT14 act as transcriptional repression motifs and are required to trigger cell death. While a single zat14 mutant does not show a cell death-related phenotype, a quintuple mutant knocking out 5 related ZAT paralogs shows a delayed onset of dPCD execution in the columella and the adjacent lateral root cap. While ZAT14 is co-expressed with established dPCD-associated genes, it does not activate their expression. Our results suggest that ZAT14 acts as a transcriptional repressor controlling a so far uncharacterized subsection of the dPCD gene regulatory network active in specific root cap tissues.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Meristem/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Fingers/physiology , Apoptosis , Gene Expression Regulation, Plant , Plant Roots/metabolismABSTRACT
Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.
Subject(s)
DNA Methylation , Endosperm , Endosperm/genetics , Endosperm/metabolism , Alleles , DNA Methylation/genetics , Genomic Imprinting/genetics , Epigenesis, Genetic , Gene Expression Regulation, PlantABSTRACT
Double fertilization in angiosperms results in the formation of a second zygote, the fertilized endosperm. Unlike its embryo sibling, the endosperm is a transient structure that eventually undergoes developmentally controlled programmed cell death (PCD) at specific time points of seed development or germination. The nature of endosperm PCD exhibits a considerable diversity, both across different angiosperm taxa and within distinct endosperm tissues. In endosperm-less species, PCD might cause central cell degeneration as a mechanism preventing the formation of a fertilized endosperm. In most other angiosperms, embryo growth necessitates the elimination of surrounding endosperm cells. Nevertheless, complete elimination of the endosperm is rare and, in most cases, specific endosperm tissues persist. In mature seeds, these persisting cells may be dead, such as the starchy endosperm in cereals, or remain alive to die only during germination, like the cereal aleurone or the endosperm of castor beans. In this review, we explore current knowledge surrounding the cellular, molecular, and genetic aspects of endosperm PCD, and the influence environmental stresses have on PCD processes. Overall, this review provides an exhaustive overview of endosperm PCD processes in angiosperms, shedding light on its diverse mechanisms and its significance in seed development and seedling establishment.
Subject(s)
Endosperm , Magnoliopsida , Endosperm/growth & development , Endosperm/physiology , Magnoliopsida/physiology , Plant Physiological Phenomena , Apoptosis , Starch , Ricinus communis , GerminationABSTRACT
Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Calcium/metabolism , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Plant Roots/metabolismABSTRACT
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.
Subject(s)
Arabidopsis/genetics , CRISPR-Cas Systems/genetics , Cloning, Molecular/methods , Gene Knockout Techniques/methods , Mutagenesis , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Genetic Vectors , Organ Specificity/genetics , Phenotype , Plant Root Cap/genetics , Plant Roots/genetics , Plant Stomata/genetics , Promoter Regions, GeneticABSTRACT
Self-incompatibility (SI) is used by many angiosperms to reject self-pollen and avoid inbreeding. In field poppy (Papaver rhoeas), SI recognition and rejection of self-pollen is facilitated by a female S-determinant, PrsS, and a male S-determinant, PrpS PrsS belongs to the cysteine-rich peptide family, whose members activate diverse signaling networks involved in plant growth, defense, and reproduction. PrsS and PrpS are tightly regulated and expressed solely in pistil and pollen cells, respectively. Interaction of cognate PrsS and PrpS triggers pollen tube growth inhibition and programmed cell death (PCD) of self-pollen. We previously demonstrated functional intergeneric transfer of PrpS and PrsS to Arabidopsis (Arabidopsis thaliana) pollen and pistil. Here, we show that PrpS and PrsS, when expressed ectopically, act as a bipartite module to trigger a self-recognition:self-destruct response in Arabidopsis independently of its reproductive context in vegetative cells. The addition of recombinant PrsS to seedling roots expressing the cognate PrpS resulted in hallmark features of the P rhoeas SI response, including S-specific growth inhibition and PCD of root cells. Moreover, inducible expression of PrsS in PrpS-expressing seedlings resulted in rapid death of the entire seedling. This demonstrates that, besides specifying SI, the bipartite PrpS-PrsS module can trigger growth arrest and cell death in vegetative cells. Heterologous, ectopic expression of a plant bipartite signaling module in plants has not been shown previously and, by extrapolation, our findings suggest that cysteine-rich peptides diversified for a variety of specialized functions, including the regulation of growth and PCD.
Subject(s)
Arabidopsis/metabolism , Apoptosis/genetics , Apoptosis/physiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Death/genetics , Cell Death/physiology , Flowers/genetics , Flowers/metabolism , Pollen/genetics , Pollen/metabolism , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Meristem/metabolism , Plant Roots/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Meristem/genetics , Plant Roots/genetics , Transcription Factors/geneticsABSTRACT
Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.
Subject(s)
Adaptation, Physiological/genetics , Arabidopsis/physiology , Plant Roots/genetics , Polyploidy , Arabidopsis/cytology , Arabidopsis/genetics , Cell Size , DNA, Plant , Gene Expression Profiling , Gene Expression Regulation, Plant , Plant Cells/physiology , Plant Roots/growth & development , Plants, Genetically Modified , Reproducibility of Results , Spatio-Temporal Analysis , Stress, Physiological/geneticsABSTRACT
Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+ We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses.
Subject(s)
Arabidopsis/drug effects , Calcium Signaling/drug effects , Indoleacetic Acids/metabolism , Nicotiana/drug effects , Small Molecule Libraries/pharmacology , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Arabidopsis/genetics , Bacterial Proteins/genetics , Bepridil/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Signaling/physiology , Cell Line , Drug Evaluation, Preclinical/methods , Fenamates/pharmacology , Indoleacetic Acids/antagonists & inhibitors , Luminescent Measurements , Luminescent Proteins/genetics , Niclosamide/pharmacology , Plant Cells/drug effects , Plant Cells/metabolism , Plant Growth Regulators/metabolism , Plant Roots/drug effects , Plants, Genetically Modified , Nicotiana/genetics , Vacuoles/drug effects , Vacuoles/metabolismABSTRACT
Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types of Aurora kinases. In plants, including Arabidopsis (Arabidopsis thaliana), these are termed α- and ß-Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α-Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS2 and 3 (TPXL2/3) prominently associated with α-Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell-cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts nonredundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cell Cycle Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Seeds/genetics , Amino Acid Sequence , Arabidopsis/embryology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Enzyme Activation/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Microscopy, Confocal , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Plants, Genetically Modified , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Proteomics/methods , Seeds/embryology , Seeds/metabolism , Sequence Homology, Amino AcidABSTRACT
Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS-PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS-green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.
Subject(s)
Arabidopsis , Papaver , Arabidopsis/genetics , Papaver/genetics , Plant Proteins , Pollen/genetics , PollinationABSTRACT
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.