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.

Root architecture and rhizosphere-microbe interactions.

Plant roots fulfil crucial tasks during a plant's life. As roots encounter very diverse conditions while exploring the soil for resources, their growth and development must be responsive to changes in the rhizosphere, resulting in root architectures that are tailor-made for all prevailing circumstances. Using multi-disciplinary approaches, we are gaining more intricate insights into the regulatory mechanisms directing root system architecture. This Special Issue provides insights into our advancement of knowledge on different aspects of root development and identifies opportunities for future research.

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.

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.

Endoreplication controls cell size via mechanochemical signaling.

During hypocotyl development, an asymmetric auxin gradient causes differential cell elongation, leading to tissue bending and apical hook formation. Recently, Ma et al. identified a molecular pathway that links auxin with endoreplication and cell size through cell wall integrity sensing, cell wall remodeling, and regulation of cell wall stiffness.

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.

Phenotypic effect of growth media on Arabidopsis thaliana root hair growth.

Over the years, many different growth media have been used to grow Arabidopsis thaliana in vitro in petri dishes. For these media the nutrient composition may vary, sugars may or may not be added, the medium may or may not be buffered and there is a choice between different gelling agents. The magnitude of possible combinations of these variables obstructs easy comparison of seedling phenotypes grown on the different media. This is especially obvious when it concerns the study of root hairs that are extremely sensitive to changes in their environment. To demonstrate this effect, we have grown Arabidopsis thaliana wild-type seeds on 18 different combinations of growth media and quantified root hair development. Comparison of root hair length and the respective root hair profiles identified the media that result in the formation of the longest root hairs. On these favored media they elongate through tip growth at a constant growth rate until they reach their final length (around 0.6 mm) at a distance of ±4 mm from the root tip.

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.

Arabidopsis root growth and development under metal exposure presented in an adverse outcome pathway framework.

Due to human activities, soils become more and more polluted with metals, which imposes risks for human health and wildlife welfare. As most of the metals end up in the food chain through accumulation in plants, we need to establish science-based environmental criteria and risk management policies. To meet these necessities, a thorough understanding is required of how these metals accumulate in and affect plants. Many studies have been conducted towards this aim, but strikingly, only a few entries can be found in ecotoxicological databases, especially on Arabidopsis thaliana, which serves as a model species for plant (cell) physiology and genetic studies. As experimental conditions seem to vary considerably throughout literature, extrapolation or comparison of data is rather difficult or should be approached with caution. Furthermore, metal-polluted soils often contain more than one metal, yet limited studies investigated the impact of metal mixtures on plants. This review aims to compile all data concerning root system architecture under Cu, Cd and Zn stress, in single or multi-metal exposure in A. thaliana, and link it to metal-induced responses at different biological levels. Global incorporation into an adverse outcome pathway framework is presented.

The quiescent centre and root apical meristem: organization and function.

This special issue is dedicated to the 100th anniversary of the birth of Frederick Albert Lionel Clowes, who discovered the quiescent centre (QC) of the root apical meristem (RAM). His discovery was a foundation for contemporary studies of the QC and RAM function, maintenance, and organization. RAM function is fundamental for cell production and root growth. This special issue bundles reviews on the main tendencies, hypotheses, and future directions, and identifies unknowns in the field.

The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators.

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

Plant biology: Positive feedback between auxin and cell wall mechanics during apical hook formation.

Apical hook formation protects fragile tissues of the hypocotyl in soil during seedling emergence. A new study reveals a positive feedback loop between asymmetric distribution of the hormone auxin and the cell wall pectin conformations underpinning cell elongation and tissue bending.

Brassinosteroids Influence Arabidopsis Hypocotyl Graviresponses through Changes in Mannans and Cellulose.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base.

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.

Osmotic stress inhibits leaf growth of Arabidopsis thaliana by enhancing ARF-mediated auxin responses.

We investigated the interaction between osmotic stress and auxin signaling in leaf growth regulation. Therefore, we grew Arabidopsis thaliana seedlings on agar media supplemented with mannitol to impose osmotic stress and 1-naphthaleneacetic acid (NAA), a synthetic auxin. We performed kinematic analysis and flow-cytometry to quantify the effects on cell division and expansion in the first leaf pair, determined the effects on auxin homeostasis and response (DR5::ß-glucuronidase), performed a next-generation sequencing transcriptome analysis and investigated the response of auxin-related mutants. Mannitol inhibited cell division and expansion. NAA increased the effect of mannitol on cell division, but ameliorated its effect on expansion. In proliferating cells, NAA and mannitol increased free IAA concentrations at the cost of conjugated IAA and stimulated DR5 promotor activity. Transcriptome analysis shows a large overlap between NAA and osmotic stress-induced changes, including upregulation of auxin synthesis, conjugation, transport and TRANSPORT INHIBITOR RESPONSE1 (TIR1) and AUXIN RESPONSE FACTOR (ARF) response genes, but downregulation of Aux/IAA response inhibitors. Consistently, arf7/19 double mutant lack the growth response to auxin and show a significantly reduced sensitivity to osmotic stress. Our results show that osmotic stress inhibits cell division during leaf growth of A. thaliana at least partly by inducing the auxin transcriptional response.

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.

The effect of metal mixture composition on toxicity to C. elegans at individual and population levels.

The toxicity of zinc (Zn), copper (Cu), and cadmium (Cd) to the nematode Caenorhabditis elegans was characterised under single metal and mixture scenarios at different organisational levels. The effects on population size and body length were investigated at two concentrations corresponding to the 24 h LC5 and LC20 levels. Metal toxicity was dependent on metal concentration, exposure time and mixture composition. Populations exposed to LC20 levels of Cd, ZnCu, CuCd and ZnCuCd plummeted, while for all LC5 concentrations, population size continued to increase, albeit that single metals were less harmful than mixtures. Combinations of the LC20 concentration of Cd with a range of Zn concentrations showed concentration dependent mitigating effects on population size and antagonistic effects on mortality. By combining effects at different organisational levels, more insight into metal toxicity was obtained. Metal effects were more evident on population size than on body length or mortality, suggesting that population size could be considered as a sensitive endpoint. Furthermore, our observations of ZnCd mixture effects at the individual and population levels are consistent with literature data on the dose-dependent expression of the cdf-2 gene, which is involved in mediation of Zn and Cd toxicity.

CBL1-CIPK26-mediated phosphorylation enhances activity of the NADPH oxidase RBOHC, but is dispensable for root hair growth.

Root hairs (RH) are tip growing polarized cells aiding the uptake of nutrients and water into plants. RH differentiation involves the interplay of various hormones and second messengers. Tightly regulated production of reactive oxygen species by the NADPH oxidase RBOHC crucially functions in RH differentiation and Ca2+ -dependent phosphorylation has been implemented in these processes. However, the kinases regulating RBOHC remained enigmatic. Here we identify CBL1-CIPK26 Ca2+ sensor-kinase complexes as modulators of RBOHC activity. Combined genetic, cell biological and biochemical analyses reveal synergistic function of CIPK26-mediated phosphorylation and Ca2+ binding for RBOHC activation. Complementation of rbohC mutant RH phenotypes by a S318/322 phosphorylation deficient RBOHC version suggests flexible and alternating phosphorylation patterns as mechanism fine-tuning ROS production in RH development.

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.