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The molecular mechanism of polar auxin transport will only be fully understood when current studies embrace the context of previous physiological experiments. Abstract Our current understanding of how plants move auxin through their tissues is largely built on the use of polar auxin transporter inhibitors. Although the most important proteins that mediate auxin transport and its regulation have probably all been identified and the mapping of their interactions is well underway, mechanistically we are still surprisingly far away from understanding how auxin is transported. Such an understanding will only emerge after new data are placed in the context of the wealth of physiological data on which they are founded. This review will look back over the use of a key inhibitor called naphthylphthalamic acid (NPA) and outline its contribution to our understanding of the molecular mechanisms of polar auxin transport, before proceeding to speculate on how its use is likely still to be informative.

A user-friendly ImageJ plugin enables the application and training of U-Nets for deep-learning-based image segmentation, detection and classification tasks with minimal labeling requirements.

The phytohormone salicylic acid (SA) is well known for its induction of pathogenesis-related proteins and systemic acquired resistance; SA also has specific effects on plant growth and development. Here we analyzed the effect of SA on Arabidopsis (Arabidopsis thaliana) root development. We show that exogenous SA treatment at low (below 50 𝜇M) and high (greater than 50 𝜇M) concentrations affect root meristem development in two different PR1-independent ways. Low-concentration SA promoted adventitious roots and altered architecture of the root apical meristem, whereas high-concentration SA inhibited all growth processes in the root. All exposures to exogenous SA led to changes in auxin synthesis and transport. A wide range of SA treatment concentrations activated auxin synthesis, but the effect of SA on auxin transport was dose dependent. Mathematical modeling of auxin synthesis and transport predicted auxin accumulation or depletion in the root tip following low- or high-concentration SA treatments, respectively. SA-induced auxin accumulation led to the formation of more layers of columella initials, an additional cortical cell layer (middle cortex), and extra files of epidermis, cortex, and endodermis cells. Suppression of SHORT ROOT and activation of CYCLIN D6;1 mediated the changes in radial architecture of the root. We propose that low-concentration SA plays an important role in shaping root meristem structure and root system architecture.

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca 2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA - triggered calcium signals, are blocked by treatment with the SCF TIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCF TIR1/AFB receptor and the CNGC14 Ca 2+ channel, mediate fast auxin signaling in roots. Auxin regulates multiple aspects of plant growth and development. Here Dindas et al. show that in root-hair cells, the AUX1 auxin influx carrier mediates proton-driven auxin import that is perceived by auxin receptors and coupled to Ca 2+ waves that may modulate adaptive responses in the root.

The histogram of oriented gradients (HOG) is widely used for image description and proves to be very effective. In many vision problems, rotation-invariant analysis is necessary or preferred. Popular solutions are mainly based on pose normalization or learning, neglecting some intrinsic properties of rotations. This paper presents a method to build rotation-invariant HOG descriptors using Fourier analysis in polar/spherical coordinates, which are closely related to the irreducible representation of the 2D/3D rotation groups. This is achieved by considering a gradient histogram as a continuous angular signal which can be well represented by the Fourier basis (2D) or spherical harmonics (3D). As rotation-invariance is established in an analytical way, we can avoid discretization artifacts and create a continuous mapping from the image to the feature space. In the experiments, we first show that our method outperforms the state-of-the-art in a public dataset for a car detection task in aerial images. We further use the Princeton Shape Benchmark and the SHREC 2009 Generic Shape Benchmark to demonstrate the high performance of our method for similarity measures of 3D shapes. Finally, we show an application on microscopic volumetric data.

Root development is regulated by the tripeptide glutathione (GSH), a strong non-enzymatic antioxidant found in plants but with a poorly understood function in roots. Here, Arabidopsis mutants deficient in GSH biosynthesis (cad2, rax1, and rml1) and plants treated with the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) showed root growth inhibition, significant alterations in the root apical meristem (RAM) structure (length and cell division), and defects in lateral root formation. Investigation of the molecular mechanisms of GSH action showed that GSH deficiency modulated total ubiquitination of proteins and inhibited the auxin-related, ubiquitination-dependent degradation of Aux/IAA proteins and the transcriptional activation of early auxin-responsive genes. However, the DR5 auxin transcriptional response differed in root apical meristem (RAM) and pericycle cells. The RAM DR5 signal was increased due to the up-regulation of the auxin biosynthesis TAA1 protein and down-regulation of PIN4 and PIN2, which can act as auxin sinks in the root tip. The transcription auxin response (the DR5 signal and expression of auxin responsive genes) in isolated roots, induced by a low (0.1 µM) auxin concentration, was blocked following GSH depletion of the roots by BSO treatment. A higher auxin concentration (0.5 µM) offset this GSH deficiency effect on DR5 expression, indicating that GSH deficiency does not completely block the transcriptional auxin response, but decreases its sensitivity. The ROS regulation of GSH, the active GSH role in cell proliferation, and GSH cross-talk with auxin assume a potential role for GSH in the modulation of root architecture under stress conditions.

Active polar transport establishes directional auxin flow and the generation of local auxin gradients implicated in plant responses and development. Auxin modulates gravitropism at the root tip and root hair morphogenesis at the differentiation zone. Genetic and biochemical analyses provide evidence for defective basipetal auxin transport in trh1 roots. The trh1, pin2, axr2 and aux1 mutants, and transgenic plants overexpressing PIN1, all showing impaired gravity response and root hair development, revealed ectopic PIN1 localization. The auxin antagonist hypaphorine blocked root hair elongation and caused moderate agravitropic root growth, also leading to PIN1 mislocalization. These results suggest that auxin imbalance leads to proximal and distal developmental defects in Arabidopsis root apex, associated with agravitropic root growth and root hair phenotype, respectively, providing evidence that these two auxin‐regulated processes are coupled. Cell‐specific subcellular localization of TRH1‐YFP in stele and epidermis supports TRH1 engagement in auxin transport, and hence impaired function in trh1 causes dual defects of auxin imbalance. The interplay between intrinsic cues determining root epidermal cell fate through the TTG/GL2 pathway and environmental cues including abiotic stresses modulates root hair morphogenesis. As a consequence of auxin imbalance in Arabidopsis root apex, ectopic PIN1 mislocalization could be a risk aversion mechanism to trigger root developmental responses ensuring root growth plasticity.

Amide-linked conjugates of indole-3-acetic acid (IAA) have been identified in most plant species. They function in storage, inactivation or inhibition of the growth regulator auxin. We investigated how the major known endogenous amide-linked IAA conjugates with auxin-like activity act in auxin signaling and what role ILR1-like proteins play in this process in Arabidopsis. We used a genetically encoded auxin sensor to show that IAA-Leu, IAA-Ala and IAA-Phe act through the TIR1-dependent signaling pathway. Furthermore, by using the sensor as a free IAA reporter, we followed conjugate hydrolysis mediated by ILR1, ILL2 and IAR3 in plant cells and correlated the activity of the hydrolases with a modulation of auxin response. The conjugate preferences that we observed are in agreement with available in vitro data for ILR1. Moreover, we identified IAA-Leu as an additional substrate for IAR3 and showed that ILL2 has a more moderate kinetic performance than observed in vitro . Finally, we proved that IAR3, ILL2 and ILR1 reside in the endoplasmic reticulum, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis which results in activation of auxin signaling.

Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.

Local accumulation of the plant growth regulator auxin mediates pattern formation in Arabidopsis roots and influences outgrowth and development of lateral root- and shoot-derived primordia. However, it has remained unclear how auxin can simultaneously regulate patterning and organ outgrowth and how its distribution is stabilized in a primordium-specific manner. Here we show that five PIN genes collectively control auxin distribution to regulate cell division and cell expansion in the primary root. Furthermore, the joint action of these genes has an important role in pattern formation by focusing the auxin maximum and restricting the expression domain of PLETHORA (PLT) genes, major determinants for root stem cell specification. In turn, PLT genes are required for PIN gene transcription to stabilize the auxin maximum at the distal root tip. Our data reveal an interaction network of auxin transport facilitators and root fate determinants that control patterning and growth of the root primordium.