Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs.

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.

Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

ROOT HAIR DEFECTIVE 2 vesicular delivery to the apical plasma membrane domain during Arabidopsis root hair development.

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

Imaging plant cells and organs with light-sheet and super-resolution microscopy.

The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.

Single Amino Acid Exchange in ACTIN2 Confers Increased Tolerance to Oxidative Stress in Arabidopsis der1-3 Mutant.

Single-point mutation in the ACTIN2 gene of the der1-3 mutant revealed that ACTIN2 is an essential actin isovariant required for root hair tip growth, and leads to shorter, thinner and more randomly oriented actin filaments in comparison to the wild-type C24 genotype. The actin cytoskeleton has been linked to plant defense against oxidative stress, but it is not clear how altered structural organization and dynamics of actin filaments may help plants to cope with oxidative stress. In this study, we characterized root growth, plant biomass, actin organization and antioxidant activity of the der1-3 mutant under oxidative stress induced by paraquat and H2O2. Under these conditions, plant growth was better in the der1-3 mutant, while the actin cytoskeleton in the der1-3 carrying pro35S::GFP:FABD2 construct showed a lower bundling rate and higher dynamicity. Biochemical analyses documented a lower degree of lipid peroxidation, and an elevated capacity to decompose superoxide and hydrogen peroxide. These results support the view that the der1-3 mutant is more resistant to oxidative stress. We propose that alterations in the actin cytoskeleton, increased sensitivity of ACTIN to reducing agent dithiothreitol (DTT), along with the increased capacity to decompose reactive oxygen species encourage the enhanced tolerance of this mutant against oxidative stress.

Actin cytoskeleton and plasma membrane aquaporins are involved in different drought response of Arabidopsis rhd2 and der1 root hair mutants.

Actin cytoskeleton and reactive oxygen species are principal determinants of root hair polarity and tip growth. Loss of function in RESPIRATORY BURST OXIDASE HOMOLOG C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2), an NADPH oxidase emitting superoxide to the apoplast, and in ACTIN 2, a vegetative actin isovariant, in rhd2-1 and der1-3 mutants, respectively, lead to similar defects in root hair formation and elongation Since early endosome-mediated polar localization of AtRBOHC/RHD2 depends on actin cytoskeleton, comparing the proteome-wide consequences of both mutations might be of eminent interest. Therefore, we employed a differential proteomic analysis of Arabidopsis rhd2-1 and der1-3 mutants. Both mutants exhibited substantial alterations in abundances of stress-related proteins. Notably, plasma membrane (PM)-localized PIP aquaporins showed contrasting abundance patterns in the mutants compared to wild-types. Drought-responsive proteins were mostly downregulated in rhd2-1 but upregulated in der1-3. Proteomic data suggest that opposite to der1-3, altered vesicular transport in rhd2-1 mutant likely contributes to the deregulation of PM-localized proteins, including PIPs. Moreover, lattice light sheet microscopy revealed reduced actin dynamics in rhd2-1 roots, a finding contrasting with previous reports on der1-3 mutant. Phenotypic experiments demonstrated a drought stress susceptibility in rhd2-1 and resistance in der1-3. Thus, mutations in AtRBOHC/RHD2 and ACTIN2 cause similar root hair defects, but they differently affect the actin cytoskeleton and vesicular transport. Reduced actin dynamics in rhd2-1 mutant is accompanied by alteration of vesicular transport proteins abundance, likely leading to altered protein delivery to PM, including aquaporins, thereby significantly affecting drought stress responses.