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.

Functions and Regulation of Programmed Cell Death in Plant Development.

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.

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.

The role of charge recombination to triplet excitons in organic solar cells.

The use of non-fullerene acceptors (NFAs) in organic solar cells has led to power conversion efficiencies as high as 18%1. However, organic solar cells are still less efficient than inorganic solar cells, which typically have power conversion efficiencies of more than 20%2. A key reason for this difference is that organic solar cells have low open-circuit voltages relative to their optical bandgaps3, owing to non-radiative recombination4. For organic solar cells to compete with inorganic solar cells in terms of efficiency, non-radiative loss pathways must be identified and suppressed. Here we show that in most organic solar cells that use NFAs, the majority of charge recombination under open-circuit conditions proceeds via the formation of non-emissive NFA triplet excitons; in the benchmark PM6:Y6 blend5, this fraction reaches 90%, reducing the open-circuit voltage by 60 mV. We prevent recombination via this non-radiative channel by engineering substantial hybridization between the NFA triplet excitons and the spin-triplet charge-transfer excitons. Modelling suggests that the rate of back charge transfer from spin-triplet charge-transfer excitons to molecular triplet excitons may be reduced by an order of magnitude, enabling re-dissociation of the spin-triplet charge-transfer exciton. We demonstrate NFA systems in which the formation of triplet excitons is suppressed. This work thus provides a design pathway for organic solar cells with power conversion efficiencies of 20% or more.

Fertility loss in senescing Arabidopsis ovules is controlled by the maternal sporophyte via a NAC transcription factor triad.

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.

KIL1 terminates fertility in maize by controlling silk senescence.

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.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

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.

Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM.

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.

Roadmap for the next decade of plant programmed cell death research.

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.

Repressive ZINC FINGER OF ARABIDOPSIS THALIANA proteins promote programmed cell death in the Arabidopsis columella root cap.

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.

Spatial and temporal regulation of parent-of-origin allelic expression in the endosperm.

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.

Endosperm cell death: roles and regulation in angiosperms.

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.

A Common C2 -Symmetric 2,2'-Biphenol Building Block and its Application in the Synthesis of (+)-di-epi-Gonytolide A.

A variety of biaryl polyketides exhibit remarkable bioactivities. However, their synthetic accessibility is often challenging. Herein, the enantioselective preparation and synthetic application of an axially chiral 2,2'-biphenol building block is outlined that represents a common motif of these intriguing natural products. Based on the highly regioselective and scalable bromination of a phenol precursor, a coupling process by Lipshutz cuprate oxidation was developed. A copper-mediated deracemization strategy proved to be superior to derivatization or kinetic resolution approaches. Key steps in the overall building block synthesis were rationalized through DFT studies. Utilizing the 2,2'-biphenol, a highly diastereoselective five step synthesis of formerly unknown (+)-di-epi-gonytolide A was developed, thus showcasing the building block's general potential for the synthesis of natural products and their derivatives. En route, the first enantioselective construction of a chromone dimer intermediate was established.

Dipole Moments and Transition Dipole Moments Calculated by Pair-Density Functional Theory with State Interaction.

We develop response-function algorithms for dipole moments and transition dipole moments for compressed multistate pair-density functional theory (CMS-PDFT). We use the method of undetermined Lagrange multipliers to derive analytical expressions and validate them using numerical differentiation. We test the accuracy of the magnitudes of predicted ground-state and excited-state dipole moments, the orientations of these dipole moments, and the orientation of transition dipole moments by comparison to experimental data. We show that CMS-PDFT has good accuracy for these quantities, and we also show that, unlike methods that neglect state interaction, CMS-PDFT yields correct behavior for the dipole moment curves in the vicinity of conical intersections. This work, therefore, opens the door to molecular dynamic simulations in strong electric fields, and we envision that CMS-PDFT can now be used to discover chemical reactions that can be controlled by an oriented external electric field upon photoexcitation of the reactants.

Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots.

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.

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.

Developing Metal-Binding Isosteres of 8-Hydroxyquinoline as Metalloenzyme Inhibitor Scaffolds.

The use of metal-binding pharmacophores (MBPs) in fragment-based drug discovery has proven effective for targeted metalloenzyme drug development. However, MBPs can still suffer from pharmacokinetic liabilities. Bioisostere replacement is an effective strategy utilized by medicinal chemists to navigate these issues during the drug development process. The quinoline pharmacophore and its bioisosteres, such as quinazoline, are important building blocks in the design of new therapeutics. More relevant to metalloenzyme inhibition, 8-hydroxyquinoline (8-HQ) and its derivatives can serve as MBPs for metalloenzyme inhibition. In this report, 8-HQ isosteres are designed and the coordination chemistry of the resulting metal-binding isosteres (MBIs) is explored using a bioinorganic model complex. In addition, the physicochemical properties and metalloenzyme inhibition activity of these MBIs were investigated to establish drug-like profiles. This report provides a new group of 8-HQ-derived MBIs that can serve as novel scaffolds for metalloenzyme inhibitor development with tunable, and potentially improved, physicochemical properties.

The effect of heavy prenatal alcohol exposure on adolescent body mass index and waist-to-height ratio at 12-13 years.

BACKGROUND: Growing evidence suggests that prenatal alcohol exposure (PAE) has the potential to impact on a wide range of physical outcomes in offspring, including metabolism and body composition, although the evidence to-date is primarily from preclinical studies. The current clinical study examined the association between heavy PAE and indirect measures of adiposity in adolescence. METHODS: Analyses drew on data from the Longitudinal Study of Australian Children, a national prospective cohort of children and their families from birth to adolescence. Participants included children with heavy PAE (≥70 g/week; n = 46), measured via maternal self-report of alcohol use during pregnancy and a comparison group of children without any PAE (n = 782), frequency matched on sex, ethnicity and socio-economic position. Body mass index (BMI) z-scores, waist-to-height ratios and proportion overweight/obese were calculated from height, weight and waist circumference measured at age 12-13 years. Two (PAE) × two (sex) ANCOVA and logistic regression models were performed, controlling for matching variables, adolescent age, pubertal status and birthweight; maternal age at birth and smoking during pregnancy. RESULTS: Female adolescents with heavy PAE during late pregnancy had significantly higher BMI z-scores (M = 0.75, SD = 0.69) and proportion overweight/obese (38.5%) than females not exposed to any prenatal alcohol (M = 0.29, SD = 1.07, P = 0.04; 23.8%, P = 0.03, respectively). There was no significant effect of heavy PAE on male adolescent BMI z-scores and proportion overweight/obese or adolescent waist-to-height ratios (all P > 0.05). CONCLUSIONS: Heavy PAE had a sex-specific effect on measures of adiposity in early adolescence, with girls more likely to have increased BMI and overweight/obesity status. Further longitudinal follow-up of children exposed to PAE is required to confirm if maternal alcohol consumption is a risk factor for later life obesity.

Ectopic Expression of a Self-Incompatibility Module Triggers Growth Arrest and Cell Death in Vegetative Cells.

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.

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.