Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development.

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.

The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

A phosphorus-limitation induced, functionally conserved DUF506 protein is a repressor of root hair elongation in plants.

Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant-microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource-use efficient and can improve soil health. While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth. Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during phosphorus (P)-limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, higher phosphate content and seedling biomass, while overexpression of the gene exhibits opposite phenotypes. Co-immunoprecipitation, pull-down and bimolecular fluorescence complementation (BiFC) analyses reveal that RXR1 physically interacts with a RabD2c GTPase in nucleus, and a rabd2c mutant phenocopies the rxr1 mutant. Furthermore, N-terminal variable region of RXR1 is crucial for inhibiting RH growth. Overexpression of a Brachypodium distachyon RXR1 homolog results in repression of RH elongation in Brachypodium. Taken together, our results reveal a novel DUF506-GTPase module with a prominent role in repression of plant RH elongation especially under P stress.

Root traits as drivers of plant and ecosystem functioning: current understanding, pitfalls and future research needs.

The effects of plants on the biosphere, atmosphere and geosphere are key determinants of terrestrial ecosystem functioning. However, despite substantial progress made regarding plant belowground components, we are still only beginning to explore the complex relationships between root traits and functions. Drawing on the literature in plant physiology, ecophysiology, ecology, agronomy and soil science, we reviewed 24 aspects of plant and ecosystem functioning and their relationships with a number of root system traits, including aspects of architecture, physiology, morphology, anatomy, chemistry, biomechanics and biotic interactions. Based on this assessment, we critically evaluated the current strengths and gaps in our knowledge, and identify future research challenges in the field of root ecology. Most importantly, we found that belowground traits with the broadest importance in plant and ecosystem functioning are not those most commonly measured. Also, the estimation of trait relative importance for functioning requires us to consider a more comprehensive range of functionally relevant traits from a diverse range of species, across environments and over time series. We also advocate that establishing causal hierarchical links among root traits will provide a hypothesis-based framework to identify the most parsimonious sets of traits with the strongest links on functions, and to link genotypes to plant and ecosystem functioning.

A starting guide to root ecology: strengthening ecological concepts and standardising root classification, sampling, processing and trait measurements.

In the context of a recent massive increase in research on plant root functions and their impact on the environment, root ecologists currently face many important challenges to keep on generating cutting-edge, meaningful and integrated knowledge. Consideration of the below-ground components in plant and ecosystem studies has been consistently called for in recent decades, but methodology is disparate and sometimes inappropriate. This handbook, based on the collective effort of a large team of experts, will improve trait comparisons across studies and integration of information across databases by providing standardised methods and controlled vocabularies. It is meant to be used not only as starting point by students and scientists who desire working on below-ground ecosystems, but also by experts for consolidating and broadening their views on multiple aspects of root ecology. Beyond the classical compilation of measurement protocols, we have synthesised recommendations from the literature to provide key background knowledge useful for: (1) defining below-ground plant entities and giving keys for their meaningful dissection, classification and naming beyond the classical fine-root vs coarse-root approach; (2) considering the specificity of root research to produce sound laboratory and field data; (3) describing typical, but overlooked steps for studying roots (e.g. root handling, cleaning and storage); and (4) gathering metadata necessary for the interpretation of results and their reuse. Most importantly, all root traits have been introduced with some degree of ecological context that will be a foundation for understanding their ecological meaning, their typical use and uncertainties, and some methodological and conceptual perspectives for future research. Considering all of this, we urge readers not to solely extract protocol recommendations for trait measurements from this work, but to take a moment to read and reflect on the extensive information contained in this broader guide to root ecology, including sections I-VII and the many introductions to each section and root trait description. Finally, it is critical to understand that a major aim of this guide is to help break down barriers between the many subdisciplines of root ecology and ecophysiology, broaden researchers' views on the multiple aspects of root study and create favourable conditions for the inception of comprehensive experiments on the role of roots in plant and ecosystem functioning.

Seedling Chloroplast Responses Induced by N-Linolenoylethanolamine Require Intact G-Protein Complexes.

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gßγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis (Arabidopsis thaliana) seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

Deletion analysis of AGD1 reveals domains crucial for plasma membrane recruitment and function in root hair polarity.

AGD1, a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP), functions in specifying root hair polarity in Arabidopsis thaliana To better understand how AGD1 modulates root hair growth, we generated full-length and domain-deleted AGD1-green fluorescent protein (GFP) constructs, and followed their localization during root hair development. AGD1-GFP localized to the cytoplasm and was recruited to specific regions of the root hair plasma membrane (PM). Distinct PM AGD1-GFP signal was first detected along the site of root hair bulge formation. The construct continued to mark the PM at the root hair apical dome, but only during periods of reduced growth. During rapid tip growth, AGD1-GFP labeled the PM of the lateral flanks and dissipated from the apical-most PM. Deletion analysis and a single domain GFP fusion revealed that the pleckstrin homology (PH) domain is the minimal unit required for recruitment of AGD1 to the PM. Our results indicate that differential recruitment of AGD1 to specific PM domains is an essential component of the membrane trafficking machinery that facilitates root hair developmental phase transitions and responses to changes in the root microenvironment.

The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis.

Symbiotic nitrogen fixation by rhizobia in legume root nodules is a key source of nitrogen for sustainable agriculture. Genetic approaches have revealed important roles for only a few of the thousands of plant genes expressed during nodule development and symbiotic nitrogen fixation. Previously, we isolated >100 nodulation and nitrogen fixation mutants from a population of Tnt1-insertion mutants of Medigaco truncatula Using Tnt1 as a tag to identify genetic lesions in these mutants, we discovered that insertions in a M. truncatula nodule-specific polycystin-1, lipoxygenase, α-toxin (PLAT) domain-encoding gene, MtNPD1, resulted in development of ineffective nodules. Early stages of nodule development and colonization by the nitrogen-fixing bacterium Sinorhizobium meliloti appeared to be normal in the npd1 mutant. However, npd1 nodules ceased to grow after a few days, resulting in abnormally small, ineffective nodules. Rhizobia that colonized developing npd1 nodules did not differentiate completely into nitrogen-fixing bacteroids and quickly degraded. MtNPD1 expression was low in roots but increased significantly in developing nodules 4 d postinoculation, and expression accompanied invading rhizobia in the nodule infection zone and into the distal nitrogen fixation zone. A functional MtNPD1:GFP fusion protein localized in the space surrounding symbiosomes in infected cells. When ectopically expressed in tobacco (Nicotiana tabacum) leaves, MtNPD1 colocalized with vacuoles and the endoplasmic reticulum. MtNPD1 belongs to a cluster of five nodule-specific single PLAT domain-encoding genes, with apparent nonredundant functions.

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis.

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of ahlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4-dichlorophenoxyacetic acid-mediated alteration of actin in Arabidopsis roots.

2,4-Dichlorophenoxyacetic acid (2,4-D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole-3-acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4-D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4-D-specific mutants suggested that 2,4-D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4-D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4-D but not IAA altered the actin structure in long-term and short-term assays. Analysis of the 2,4-D-specific mutant aar1-1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4-D-induced depolymerization of actin. The ubiquitin proteasome mutants tir1-1 and axr1-12, which show enhanced resistance to 2,4-D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4-D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4-D on the organization of actin filaments. Roots of the double mutant aar1-1 tir1-1 also showed enhanced resistance to 2,4-D-induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4-D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCFTIR1 ubiquitin proteasome components.

Two Chloroplast-Localized Proteins: AtNHR2A and AtNHR2B, Contribute to Callose Deposition During Nonhost Disease Resistance in Arabidopsis.

Plants are naturally resistant to most pathogens through a broad and durable defense response called nonhost disease resistance. Nonhost disease resistance is a complex process that includes preformed physical and chemical barriers and induced responses. In spite of its importance, many components of nonhost disease resistance remain to be identified and characterized. Using virus-induced gene silencing in Nicotiana benthamiana, we discovered a novel gene that we named NbNHR2 (N. benthamiana nonhost resistance 2). NbNHR2-silenced plants were susceptible to the nonadapted pathogen Pseudomonas syringae pv. tomato T1, which does not cause disease in wild-type or nonsilenced N. benthamiana plants. We found two orthologous genes in Arabidopsis thaliana: AtNHR2A and AtNHR2B. Similar to the results obtained in N. benthamiana, Atnhr2a and Atnhr2b mutants were susceptible to the nonadapted bacterial pathogen of A. thaliana, P. syringae pv. tabaci. We further found that these mutants were also defective in callose deposition. AtNHR2A and AtNHR2B fluorescent protein fusions transiently expressed in N. benthamiana localized predominantly to chloroplasts and a few unidentified dynamic puncta. RFP-AtNHR2A and AtNHR2B-GFP displayed overlapping signals in chloroplasts, indicating that the two proteins could interact, an idea supported by coimmunoprecipitation studies. We propose that AtNHR2A and AtNHR2B are new components of a chloroplast-signaling pathway that activates callose deposition to the cell wall in response to bacterial pathogens.

Malonylation of Glucosylated N-Lauroylethanolamine: A NEW PATHWAY THAT DETERMINES N-ACYLETHANOLAMINE METABOLIC FATE IN PLANTS.

N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives present in trace amounts in many eukaryotes. Although NAEs have signaling and physiological roles in animals, little is known about their metabolic fate in plants. Our previous microarray analyses showed that inhibition of Arabidopsis thaliana seedling growth by exogenous N-lauroylethanolamine (NAE 12:0) was accompanied by the differential expression of multiple genes encoding small molecule-modifying enzymes. We focused on the gene At5g39050, which encodes a phenolic glucoside malonyltransferase 1 (PMAT1), to better understand the biological significance of NAE 12:0-induced gene expression changes. PMAT1 expression was induced 3-5-fold by exogenous NAE 12:0. PMAT1 knockouts (pmat1) had reduced sensitivity to the growth-inhibitory effects of NAE 12:0 compared with wild type leading to the hypothesis that PMAT1 might be a previously uncharacterized regulator of NAE metabolism in plants. To test this hypothesis, metabolic profiling of wild-type and pmat1 seedlings treated with NAE 12:0 was conducted. Wild-type seedlings treated with NAE 12:0 accumulated glucosylated and malonylated forms of this NAE species, and structures were confirmed using nuclear magnetic resonance (NMR) spectroscopy. By contrast, only the peak corresponding to NAE 12:0-glucoside was detected in pmat1 Recombinant PMAT1 catalyzed the reaction converting NAE 12:0-glucoside to NAE 12:0-mono- or -dimalonylglucosides providing direct evidence that this enzyme is involved in NAE 12:0-glucose malonylation. Taken together, our results indicate that glucosylation of NAE 12:0 by a yet to be determined glucosyltransferase and its subsequent malonylation by PMAT1 could represent a mechanism for modulating the biological activities of NAEs in plants.

Lipoxygenase-derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.

Ethanolamide-conjugated fatty acid derivatives, also known as N-acylethanolamines (NAEs), occur at low levels (µg per g) in desiccated seeds, and endogenous amounts decline rapidly with seedling growth. Linoleoylethanolamide (NAE18:2) is the most abundant of these NAEs in seeds of almost all plants, including Arabidopsis thaliana. In Arabidopsis, NAE18:2 may be oxidized by lipoxygenase (LOX) or hydrolyzed by fatty acid amide hydrolase (FAAH) during normal seedling establishment, and this contributes to the normal progression of NAE depletion that is coincident with the depletion of abscisic acid (ABA). Here we provide biochemical, genetic and pharmacological evidence that a specific 9-LOX metabolite of NAE18:2 [9-hydro(pero)xy linoleoylethanolamide (9-NAE-H(P)OD)] has a potent negative influence on seedling root elongation, and acts synergistically with ABA to modulate the transition from embryo to seedling growth. Genetic analyses using mutants in ABA synthesis (aba1 and aba2), perception (pyr1, pyl1, pyl2, pyl4, pyl5 and pyl8) or transcriptional activation (abi3-1) indicated that arrest of root growth by 9-NAE-H(P)OD requires an intact ABA signaling pathway, and probably operates to increase ABA synthesis as part of a positive feedback loop to modulate seedling establishment in response to adverse environmental conditions. These results identify a specific, bioactive ethanolamide oxylipin metabolite of NAE18:2, different from those of ethanolamide-conjugated linolenic acid (NAE18:3), as well as a molecular explanation for its inhibitory action, emphasizing the oxidative metabolism of NAEs as an important feature of seedling development.

Ethanolamide oxylipins of linolenic acid can negatively regulate Arabidopsis seedling development.

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.

Synthesis of phenoxyacyl-ethanolamides and their effects on fatty acid amide hydrolase activity.

N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals.

N-Acylethanolamines: lipid metabolites with functions in plant growth and development.

Twenty years ago, N-acylethanolamines (NAEs) were considered by many lipid chemists to be biological 'artifacts' of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N-arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross-kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.

Transcriptional response of Arabidopsis seedlings during spaceflight reveals peroxidase and cell wall remodeling genes associated with root hair development.

UNLABELLED: • PREMISE OF THE STUDY: Plants will be an important component of advanced life support systems during space exploration missions. Therefore, understanding their biology in the spacecraft environment will be essential before they can be used for such systems.• METHODS: Seedlings of Arabidopsis thaliana were grown for 2 wk in the Biological Research in Canisters (BRIC) hardware on board the second to the last mission of the space shuttle Discovery (STS-131). Transcript profiles between ground controls and space-grown seedlings were compared using stringent selection criteria.• KEY RESULTS: Expression of transcripts associated with oxidative stress and cell wall remodeling was repressed in microgravity. These downregulated genes were previously shown to be enriched in root hairs consistent with seedling phenotypes observed in space. Mutations in genes that were downregulated in microgravity, including two uncharacterized root hair-expressed class III peroxidase genes (PRX44 and PRX57), led to defective polar root hair growth on Earth. PRX44 and PRX57 mutants had ruptured root hairs, which is a typical phenotype of tip-growing cells with defective cell walls and those subjected to stress.• CONCLUSIONS: Long-term exposure to microgravity negatively impacts tip growth by repressing expression of genes essential for normal root hair development. Whereas changes in peroxidase gene expression leading to reduced root hair growth in space are actin-independent, root hair development modulated by phosphoinositides could be dependent on the actin cytoskeleton. These results have profound implications for plant adaptation to microgravity given the importance of tip growing cells such as root hairs for efficient nutrient capture.