MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots.

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.

The Proline-Rich Family Protein EXTENSIN33 Is Required for Etiolated Arabidopsis thaliana Hypocotyl Growth.

Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.

Hormonal regulation of root hair growth and responses to the environment in Arabidopsis.

The main functions of plant roots are water and nutrient uptake, soil anchorage, and interaction with soil-living biota. Root hairs, single cell tubular extensions of root epidermal cells, facilitate or enhance these functions by drastically enlarging the absorptive surface. Root hair development is constantly adapted to changes in the root's surroundings, allowing for optimization of root functionality in heterogeneous soil environments. The underlying molecular pathway is the result of a complex interplay between position-dependent signalling and feedback loops. Phytohormone signalling interconnects this root hair signalling cascade with biotic and abiotic changes in the rhizosphere, enabling dynamic hormone-driven changes in root hair growth, density, length, and morphology. This review critically discusses the influence of the major plant hormones on root hair development, and how changes in rhizosphere properties impact on the latter.

Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly.

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.

In vivo measurement of the Young's modulus of the cell wall of single root hairs.

Root hairs are cells from the root epidermis that grow as long tubular bulges perpendicular to the root. They can grow in a variety of mechanical or chemical environments. Their mechanical properties are mainly due to their stiff cell wall which also constitutes a physical barrier between the cell and its environment. Thus, it is essential to be able to quantify the cell wall mechanical properties and their adaptation to environmental cues. Here, we present a technique we developed to measure the Young's (elastic) modulus of the root hair cell wall. In essence, using custom-made glass microplates as cantilevers of calibrated stiffness, we are able to measure the force necessary to bend a single living root hair. From these experiments one can determine the stiffness and Young's modulus of the root hair cell wall.

Accumulation and effects of perfluoroalkyl substances in Arabidopsis thaliana in a temperature-dependent manner: an in vitro study.

The replacement of long-chained per- and polyfluoroalkyl substances (PFAS) with their short-chained homologues may have an impact on the accumulation in plants. The extent to which PFAS are absorbed by plants may differ among species and may depend on environmental factors, including temperature. The effect of an increased temperature on root uptake and translocation of PFAS in plants has been poorly studied. In addition, very few studies have examined toxicity of environmentally realistic PFAS concentrations to plants. Here, we investigated the bioaccumulation and tissue-distribution of fifteen PFAS in Arabidopsis thaliana L. grown in vitro at two different temperatures. Additionally, we examined the combined effects of temperature and PFAS accumulation on plant growth. Short-chained PFAS mainly accumulated in the leaves. The perfluorocarboxylic acid (PFCA) concentrations in roots and leaves, and the relative contribution of PFCAs to the ΣPFAS concentrations increased with carbon chain length regardless of temperature, with the exception of perfluorobutanoic acid (PFBA). An increased uptake of PFAS in leaves and roots at higher temperatures was observed for PFAS containing either eight or nine carbon atoms and could hence potentially result in higher risks for human intake. Leaf:root ratios of PFCAs followed a U-shaped pattern with carbon chain length, which is attributed to both hydrophobicity and anion exchange. Overall, no combined effects of realistic PFAS concentrations and temperature on the growth of A. thaliana were observed. PFAS exposure positively affected early root growth rates and root hair lengths, indicating a potential effect on factors involved in root hair morphogenesis. However, this effect on root growth rate became negligible later on in the exposure, and solely a temperature effect was observed after 6 days. Temperature also affected the leaf surface area. The underlying mechanisms on how PFAS stimulates root hair growth require further examination.

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Shank-localized cell wall growth contributes to Arabidopsis root hair elongation.

Root hairs are highly elongated tubular extensions of root epidermal cells with a plethora of physiological functions, particularly in establishing the root-rhizosphere interface. Anisotropic expansion of root hairs is generally thought to be exclusively mediated by tip growth-a highly controlled apically localized secretion of cell wall material-enriched vesicles that drives the extension of the apical dome. Here we show that tip growth is not the only mode of root hair elongation. We identified events of substantial shank-localized cell wall expansion along the polar growth axis of Arabidopsis root hairs using morphometric analysis with quantum dots. These regions expanded after in vivo immunolocalization using cell wall-directed antibodies and appeared as distinct bands that were devoid of cell wall labelling. Application of a novel click chemistry-enabled galactose analogue for pulse chase and real-time imaging allowed us to label xyloglucan, a major root hair glycan, and demonstrate its de novo deposition and enzymatic remodelling in these shank regions. Our data reveal a previously unknown aspect of root hair growth in which both tip- and shank-localized dynamic cell wall deposition and remodelling contribute to root hair elongation.

The differential tolerance of C3 and C4 cereals to aluminum toxicity is faded under future CO2 climate.

Industrial activities have led to a gradual and global increase in soil aluminum (Al) and atmospheric CO2 concentrations. Al bioavailability strongly depends on the soil pH, which in turn is affected by atmospheric CO2 levels. In spite of the concurrent impact which Al and elevated CO2 (eCO2) could have on plants, their interaction and how it might affect the growth of economically important crop species has not been investigated. Here, we have investigated the combined impact of soil Al and eCO2 exposure on key C3 (wheat, oat) and C4 (maize, sorghum) crops, at the physiological and biochemical level. Compared to C3 plants, C4 plants accumulated less Al by stimulating soil Al retention through exudation of root organic acids. Consequently, Al-exposed C4 plants maintained photosynthetic performance and anti-oxidative capacity. Exposure to eCO2 reduced the stress responses of C3 and C4 crops to Al exposure. Elevated CO2 decreased Al accumulation and oxidative damage in all cereals, and ameliorated C3 plant growth. This was reflected on the biochemical level, where eCO2 inhibited ROS production and restored RuBisCo activity in C3 crops only. Overall, our data suggest that, compared to C3 crops, C4 cereals are more tolerant to soil Al exposure under current ambient CO2 (aCO2) levels whereas future eCO2 levels might stimulate Al tolerance in C3 crops.

An actinomycete strain of Nocardiopsis lucentensis reduces arsenic toxicity in barley and maize.

Accumulation of arsenic in plant tissues poses a substantial threat to global crop yields. The use of plant growth-promoting bacterial strains to mitigate heavy metal toxicity has been illustrated before. However, its potential to reduce plant arsenic uptake and toxicity has not been investigated to date. Here, we describe the identification and characterization of a Nocardiopsis lucentensis strain isolated from heavy metal contaminated soil. Inoculation with this bioactive actinomycete strain decreased arsenic root and shoot bioaccumulation in both C3 and C4 crop species namely barley and maize. Upon arsenate treatment, N. lucentensis S5 stimulated root citric acid production and the plant's innate detoxification capacity in a species-specific manner. In addition, this specific strain promoted biomass gain, despite substantial tissue arsenic levels. Detoxification (metallothionein, phytochelatin, glutathione-S-transferase levels) was upregulated in arsenate-exposed shoot and roots, and this response was further enhanced upon S5 supplementation, particularly in barley and maize roots. Compared to barley, maize plants were more tolerant to arsenate-induced oxidative stress (less H2O2 and lipid peroxidation levels). However, barley plants invested more in antioxidative capacity induction (ascorbate-glutathione turnover) to mitigate arsenic oxidative stress, which was strongly enhanced by S5. We quantify and mechanistically discuss the physiological and biochemical basis of N. lucentensis-mediated plant biomass recovery on arsenate polluted soils. Our findings substantiate the potential applicability of a bactoremediation strategy to mitigate arsenic-induced yield loss in crops.

Soil arsenic toxicity differentially impacts C3 (barley) and C4 (maize) crops under future climate atmospheric CO2.

Soil arsenic (As) contamination limits global agricultural productivity. Anthropogenic emissions are causing atmospheric CO2 levels to rise. Elevated CO2 (eCO2) boosts plant growth both under optimal and suboptimal growth conditions. However, the crop-specific interaction between eCO2 and soil arsenic exposure has not been investigated at the whole plant, physiological and biochemical level. Here, we tested the effects of eCO2 (620 ppm) and soil As exposure (mild and severe treatments, 25 and 100 mg As/Kg soil) on growth, photosynthesis and redox homeostasis in barley (C3) and maize (C4). Compared to maize, barley was more susceptible to soil As exposure at ambient CO2 levels. Barley plants accumulated more As, particularly in roots. As accumulation inhibited plant growth and induced oxidative damage in a species-specific manner. As-exposed barley experienced severe oxidative stress as illustrated by high H2O2 and protein oxidation levels. Interestingly, eCO2 differentially mitigated As-induced stress in barley and maize. In barley, eCO2 exposure reduced photorespiration, H2O2 production, and lipid/protein oxidation. In maize eCO2 exposure led to an upregulation of the ascorbate-glutathione (ASC/GSH)-mediated antioxidative defense system. Combined, this work highlights how ambient and future eCO2 levels differentially affect the growth, physiology and biochemistry of barley and maize crops exposed to soil As pollution.

The Auxin-Regulated CrRLK1L Kinase ERULUS Controls Cell Wall Composition during Root Hair Tip Growth.

Root hairs facilitate a plant's ability to acquire soil anchorage and nutrients. Root hair growth is regulated by the plant hormone auxin and dependent on localized synthesis, secretion, and modification of the root hair tip cell wall. However, the exact cell wall regulators in root hairs controlled by auxin have yet to be determined. In this study, we describe the characterization of ERULUS (ERU), an auxin-induced Arabidopsis receptor-like kinase, whose expression is directly regulated by ARF7 and ARF19 transcription factors. ERU belongs to the Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) subfamily of putative cell wall sensor proteins. Imaging of a fluorescent fusion protein revealed that ERU is localized to the apical root hair plasma membrane. ERU regulates cell wall composition in root hairs and modulates pectin dynamics through negative control of pectin methylesterase (PME) activity. Mutant eru (-/-) root hairs accumulate de-esterified homogalacturonan and exhibit aberrant pectin Ca2+-binding site oscillations and increased PME activity. Up to 80% of the eru root hair phenotype is rescued by pharmacological supplementation with a PME-inhibiting catechin extract. ERU transcription is altered in specific cell wall-related root hair mutants, suggesting that it is a target for feedback regulation. Loss of ERU alters the phosphorylation status of FERONIA and H+-ATPases 1/2, regulators of apoplastic pH. Furthermore, H+-ATPases 1/2 and ERU are differentially phosphorylated in response to auxin. We conclude that ERULUS is a key auxin-controlled regulator of cell wall composition and pectin dynamics during root hair tip growth.

The Kinase ERULUS Controls Pollen Tube Targeting and Growth in Arabidopsis thaliana.

In this paper, we describe the role of the receptor-like kinase ERULUS (ERU) in PT growth of Arabidopsis thaliana. In silico analysis and transcriptional reporter lines revealed that ERU is only expressed in pollen and root hairs (RHs), making it a tip growth-specific kinase. Deviations from Mendelian inheritance were observed in the offspring of self-pollinated heterozygous eru plants. We found that in vivo eru PT targeting was disturbed, providing a possible explanation for the observed decrease in eru fertilization competitiveness. Extracellular calcium perception and intracellular calcium dynamics lie at the basis of in vivo pollen tube (PT) tip growth and guidance. In vitro, ERU loss-of-function lines displayed no obvious PT phenotype, unless grown on low extracellular calcium ([Ca2+]ext) medium. When grown at 12 the normal [Ca2+]ext, eru PTs grew 37% slower relative to WT PTs. Visualization of cytoplasmic [Ca2+]cyt oscillations using the Yellow Cameleon 3.6 (YC3.6) calcium sensor showed that, unlike in WT PTs, eru apical [Ca2+]cyt oscillations occur at a lower frequency when grown at lower [Ca2+]ext, consistent with the observed reduced growth velocity. Our results show that the tip growth-specific kinase ERULUS is involved in regulating Ca2+-dependent PT growth, and most importantly, fertilization efficiency through successful PT targeting to the ovules.

Perturbation of Auxin Homeostasis and Signaling by PINOID Overexpression Induces Stress Responses in Arabidopsis.

Under normal and stress conditions plant growth require a complex interplay between phytohormones and reactive oxygen species (ROS). However, details of the nature of this crosstalk remain elusive. Here, we demonstrate that PINOID (PID), a serine threonine kinase of the AGC kinase family, perturbs auxin homeostasis, which in turn modulates rosette growth and induces stress responses in Arabidopsis plants. Arabidopsis mutants and transgenic plants with altered PID expression were used to study the effect on auxin levels and stress-related responses. In the leaves of plants with ectopic PID expression an accumulation of auxin, oxidative burst and disruption of hormonal balance was apparent. Furthermore, PID overexpression led to the accumulation of antioxidant metabolites, while pid knockout mutants showed only moderate changes in stress-related metabolites. These physiological changes in the plants overexpressing PID modulated their response toward external drought and osmotic stress treatments when compared to the wild type. Based on the morphological, transcriptome, and metabolite results, we propose that perturbations in the auxin hormone levels caused by PID overexpression, along with other hormones and ROS downstream, cause antioxidant accumulation and modify growth and stress responses in Arabidopsis. Our data provide further proof for a strong correlation between auxin and stress biology.

Prioritization of contaminated watercourses using an integrated biomarker approach in caged carp.

Because of the ever increasing complexity of environmental contamination profiles, there are limitations to the use of analytical pollutant measurements for monitoring and prioritization of watercourses. The potential of biomarkers has been debated for many years, especially in laboratory settings, but there is a need for studies evaluating these approaches in the field. We evaluated the usefulness of a selection of biomarkers, mostly indicators of general physiological status and common stress responses such as oxidative stress, to discriminate among environmental pollution profiles, with the aim of prioritizing contaminated watercourses for targeted remediation efforts. To this end, juvenile common carp (Cyprinus carpio Lin.) were exposed in cages in the field to Flemish watercourses with varying pollution profiles. After six weeks of exposure, the bioaccumulation of key pollutants was measured, and a set of organismal, biochemical and transcriptional endpoints was determined in several tissue types. After data integration a discrete set of 14 parameters was identified, that could successfully distinguish all watercourses from each other. We show that an integrated biomarker approach, mainly targeting common stress responses, can offer the resolving power to discriminate among environmentally relevant exposure scenarios, and a means to prioritize watercourses for targeted remediation.

Cell Fate Determination and the Switch from Diffuse Growth to Planar Polarity in Arabidopsis Root Epidermal Cells.

Plant roots fulfill important functions as they serve in water and nutrient uptake, provide anchorage of the plant body in the soil and in some species form the site of symbiotic interactions with soil-living biota. Root hairs, tubular-shaped outgrowths of specific epidermal cells, significantly increase the root's surface area and aid in these processes. In this review we focus on the molecular mechanisms that determine the hair and non-hair cell fate of epidermal cells and that define the site on the epidermal cell where the root hair will be initiated (=planar polarity determination). In the model plant Arabidopsis, trichoblast and atrichoblast cell fate results from intra- and intercellular position-dependent signaling and from complex feedback loops that ultimately regulate GL2 expressing and non-expressing cells. When epidermal cells reach the end of the root expansion zone, root hair promoting transcription factors dictate the establishment of polarity within epidermal cells followed by the selection of the root hair initiation site at the more basal part of the trichoblast. Molecular players in the abovementioned processes as well as the role of phytohormones are discussed, and open areas for future experiments are identified.