Respiratory Burst Oxidase Homologues (RBOHs) are involved in plant growth, development, and stress adaptation. How OsRBOHs affect root hair formation and consequently nutrient acquisition and drought resistance in rice is not well understood. We knocked out six OsRBOH genes in rice that were expressed in roots and identified OsRBOHE as the only one affecting root hair formation. OsRBOHE was strongly expressed in the root epidermis, root hairs and tiller buds. OsRBOHE is localised at the plasma membrane. Knockout of OsRBOHE decreased reactive oxygen species generation in the root hairs and tiller buds, downregulated genes involved in cell wall biogenesis, and decreased root hair length and tillering by 90% and 30%, respectively. Knockout of OsRBOHE decreased phosphorus acquisition only in low available P soil under aerobic conditions, but not in high P soil or under flooded conditions when P was likely not limited by diffusion. Knockout of OsRBOHE markedly decreased drought resistance of rice plants through the effect on root hair formation and the associated rhizosheath. Taken together, OsRBOHE is crucial for root hair formation and tillering and consequently on drought resistance in rice. The contribution of root hairs to P acquisition in rice is limited to aerobic soil.

Metabolic factors are essential for developmental biology of an organism. In plants, roots fulfill important functions, in part due to the development of specific epidermal cells, called hair cells that form root hairs (RHs) responsible for water and mineral uptake. RH development consists in (a) patterning processes involved in formation of hair and non-hair cells developed from trichoblasts and atrichoblasts; (b) RH initiation; and (c) apical (tip) growth of the RH. Here we review how these processes depend on pools of different amino acids and what is known about RH phenotypes of mutants disrupted in amino acid biosynthesis. This analysis shows that some amino acids, particularly aromatic ones, are required for RH apical (tip) growth, and that not much is known about the role of amino acids at earlier stages of RH formation. We also address the role of amino acids in rhizosphere, inhibitory and stimulating effects of amino acids on RH growth, amino acids as N source in plant nutrition, and amino acid transporters and their expression in the RHs. Amino acids form conjugates with auxin, a hormone essential for RH growth, and respective genes are overviewed. Finally, we outline missing links and envision some perspectives in the field.

Population expansion is a global issue, especially for food production. Meanwhile, global climate change is damaging our soils, making it difficult for crops to thrive and lowering both production and quality. Poor nutrition and salinity stress affect plant growth and development. Although the impact of individual plant stresses has been studied for decades, the real stress scenario is more complex due to the exposure to multiple stresses at the same time. Here we investigate using existing evidence and a meta-analysis approach to determine molecular linkages between two contemporaneous abiotic stimuli, phosphate (Pi) deficiency and salinity, on a single plant cell model, the root hairs (RHs), which is the first plant cell exposed to them. Understanding how these two stresses work molecularly in RHs may help us build super-adaptable crops and sustainable agriculture in the face of global climate change.

Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools. Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition. We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.

Plant growth-promoting microbes (PGPM) can enhance crop yield and health, but knowledge of their mode-of-action is limited. We studied the influence of two Bacillus subtilis strains, the natural isolate ALC_02 and the domesticated 168 Gö, on Arabidopsis and hypothesized that they modify the root architecture by modulating hormone transport or signaling. Both bacteria promoted increase of shoot and root surface area in vitro, but through different root anatomical traits. Mutant plants deficient in auxin transport or signaling responded less to the bacterial strains than the wild-type, and application of the auxin transport inhibitor NPA strongly reduced the influence of the strains. Both bacteria produced auxin and enhanced shoot auxin levels in DR5::GUS reporter plants. Accordingly, most of the beneficial effects of the strains were dependent on functional auxin transport and signaling, while only 168 Gö depended on functional ethylene signaling. As expected, only ALC_02 stimulated plant growth in soil, unlike 168 Gö that was previously reported to have reduced biofilms. Collectively, the results highlight that B. subtilis strains can have strikingly different plant growth-promoting properties, dependent on what experimental setup they are tested in, and the importance of choosing the right PGPM for a desired root phenotype.

Root hairs have become an important model system for studying plant growth, and in particular how plants modulate their growth in response to cell-intrinsic and environmental stimuli. In this review, we discuss recent advances in our understanding of the molecular mechanisms underlying the growth of Arabidopsis root hairs in the interface between responses to environmental cues (e.g. nutrients such as nitrates and phosphate, and microorganisms) and hormonal stimuli (e.g. auxin). Growth of root hairs is under the control of several transcription factors that are also under strong regulation at different levels. We highlight recent new discoveries along these transcriptional pathways that might have the potential to increase our capacity to enhance nutrient uptake by the roots in the context of abiotic stresses. We use the text-mining capacities of the PlantConnectome database to generate an up-to-date view of root hairs growth within these complex biological contexts.

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Deciphering root exudate composition of soil-grown plants is considered a crucial step to better understand plant-soil-microbe interactions affecting plant growth performance. In this study, two genotypes of Zea mays L. (WT, rth3) differing in root hair elongation were grown in the field in two substrates (sand, loam) in custom-made, perforated columns inserted into the field plots. Root exudates were collected at different plant developmental stages (BBCH 14, 19, 59, 83) using a soil-hydroponic-hybrid exudation sampling approach. Exudates were characterized by LC-MS based non-targeted metabolomics, as well as by photometric assays targeting total dissolved organic carbon, soluble carbohydrates, proteins, amino acids, and phenolics. Results showed that plant developmental stage was the main driver shaping both the composition and quantity of exuded compounds. Carbon (C) exudation per plant increased with increasing biomass production over time, while C exudation rate per cm² root surface area h-1 decreased with plant maturity. Furthermore, exudation rates were higher in the substrate with lower nutrient mobility (i.e., loam). Surprisingly, we observed higher exudation rates in the root hairless rth3 mutant compared to the root hair-forming WT sibling, though exudate metabolite composition remained similar. Our results highlight the impact of plant developmental stage on the plant-soil-microbe interplay.

Legumes enter into symbiotic associations with soil nitrogen-fixing rhizobia, culminating in the creation of new organs, root nodules. This complex process relies on chemical and physical interaction between legumes and rhizobia, including early signalling events informing the host legume plant of a potentially beneficial microbe and triggering the nodulation program. The great significance of this plant-microbe interaction rests upon conversion of atmospheric dinitrogen not accessible to plants into a biologically active form of ammonia available to plants. The plant cytoskeleton consists in a highly dynamic network and undergoes rapid remodelling upon sensing various developmental and environmental cues, including response to attachment, internalization, and accommodation of rhizobia in plant root and nodule cells. This dynamic nature is governed by cytoskeleton-associated proteins that modulate cytoskeletal behaviour depending on signal perception and transduction. Precisely localized cytoskeletal rearrangements are therefore essential for the uptake of rhizobia, their targeted delivery, and establishing beneficial root nodule symbiosis. This review summarizes current knowledge about rhizobia-dependent rearrangements and functions of the cytoskeleton in legume roots and nodules. General patterns and nodule type-, nodule stage-, and species-specific aspects of actin filaments and microtubules remodelling are discussed. Moreover, emerging evidence is provided about fine-tuning the root nodulation process through cytoskeleton-associated proteins. We also consider future perspectives on dynamic localization studies of the cytoskeleton during early symbiosis utilizing state of the art molecular and advanced microscopy approaches. Based on acquired detailed knowledge of the mutualistic interactions with microbes, these approaches could contribute to broader biotechnological crop improvement.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.