Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Author Correction: U-Net: deep learning for cell counting, detection, and morphometry.

In the version of this paper originally published, one of the affiliations for Dominic Mai was incorrect: "Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany" should have been "Life Imaging Center, Center for Biological Systems Analysis, Albert-Ludwigs-University, Freiburg, Germany." This change required some renumbering of subsequent author affiliations. These corrections have been made in the PDF and HTML versions of the article, as well as in any cover sheets for associated Supplementary Information.

U-Net: deep learning for cell counting, detection, and morphometry.

U-Net is a generic deep-learning solution for frequently occurring quantification tasks such as cell detection and shape measurements in biomedical image data. We present an ImageJ plugin that enables non-machine-learning experts to analyze their data with U-Net on either a local computer or a remote server/cloud service. The plugin comes with pretrained models for single-cell segmentation and allows for U-Net to be adapted to new tasks on the basis of a few annotated samples.

A method for characterizing phenotypic changes in highly variable cell populations and its application to high content screening of Arabidopsis thaliana protoplasts.

Quantitative image analysis procedures are necessary for the automated discovery of effects of drug treatment in large collections of fluorescent micrographs. When compared to their mammalian counterparts, the effects of drug conditions on protein localization in plant species are poorly understood and underexplored. To investigate this relationship, we generated a large collection of images of single plant cells after various drug treatments. For this, protoplasts were isolated from six transgenic lines of A. thaliana expressing fluorescently tagged proteins. Eight drugs at three concentrations were applied to protoplast cultures followed by automated image acquisition. For image analysis, we developed a cell segmentation protocol for detecting drug effects using a Hough transform-based region of interest detector and a novel cross-channel texture feature descriptor. In order to determine treatment effects, we summarized differences between treated and untreated experiments with an L1 Cramér-von Mises statistic. The distribution of these statistics across all pairs of treated and untreated replicates was compared to the variation within control replicates to determine the statistical significance of observed effects. Using this pipeline, we report the dose dependent drug effects in the first high-content Arabidopsis thaliana drug screen of its kind. These results can function as a baseline for comparison to other protein organization modeling approaches in plant cells. © 2017 International Society for Advancement of Cytometry.

The iRoCS Toolbox--3D analysis of the plant root apical meristem at cellular resolution.

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three-dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single-cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open-source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem.

The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis.

The plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin-dependent transcriptional activity.

The Arabidopsis thaliana Mob1A gene is required for organ growth and correct tissue patterning of the root tip.

BACKGROUND AND AIMS: The Mob1 family includes a group of kinase regulators conserved throughout eukaryotes. In multicellular organisms, Mob1 is involved in cell proliferation and apoptosis, thus controlling appropriate cell number and organ size. These functions are also of great importance for plants, which employ co-ordinated growth processes to explore the surrounding environment and respond to changing external conditions. Therefore, this study set out to investigate the role of two Arabidopsis thaliana Mob1-like genes, namely Mob1A and Mob1B, in plant development. METHODS: A detailed spatio-temporal analysis of Mob1A and Mob1B gene expression was performed by means of bioinformatic tools, the generation of expression reporter lines and in situ hybridization of gene-specific probes. To explore the function of the two genes in plant development, knock-out and knock-down mutants were isolated and their phenotype quantitatively characterized. KEY RESULTS: Transcripts of the two genes were detected in specific sets of cells in all plant organs. Mob1A was upregulated by several stress conditions as well as by abscisic acid and salicylic acid. A knock-out mutation in Mob1B did not cause any visible defect in plant development, whereas suppression of Mob1A expression affected organ growth and reproduction. In the primary root, reduced levels of Mob1A expression brought about severe defects in tissue patterning of the stem cell niche and columella and led to a decrease in meristem size. Moreover, loss of Mob1A function resulted in a higher sensitivity of root growth to abscisic acid. CONCLUSIONS: Taken together, the results indicate that arabidopsis Mob1A is involved in the co-ordination of tissue patterning and organ growth, similarly to its orthologues in other multicellular eukaryotes. In addition, Mob1A serves a plant-specific function by contributing to growth adjustments in response to stress conditions.

A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors.

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake.

In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells.

Plant Signaling: HY5 Synchronizes Resource Supply.

A new report shows that the HY5 transcription factor moves from shoots to roots in plants, mediating light regulation of root growth and nitrate uptake. This finding offers not only a mechanistic insight into shoot-root communication, but also scope for increasing crop yields.

Hydrolases of the ILR1-like family of Arabidopsis thaliana modulate auxin response by regulating auxin homeostasis in the endoplasmic reticulum.

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.

A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics.

Time-resolved quantitative analysis of auxin-mediated processes in plant cells is as of yet limited. By applying a synergistic mammalian and plant synthetic biology approach, we have developed a novel ratiometric luminescent biosensor with wide applicability in the study of auxin metabolism, transport, and signalling. The sensitivity and kinetic properties of our genetically encoded biosensor open new perspectives for the analysis of highly complex auxin dynamics in plant growth and development.

Intracellular auxin transport in pollen: PIN8, PIN5 and PILS5.

Cellular auxin homeostasis is controlled at many levels that include auxin biosynthesis, auxin metabolism, and auxin transport. In addition to intercellular auxin transport, auxin homeostasis is modulated by auxin flow through the endoplasmic reticulum (ER). PIN5, a member of the auxin efflux facilitators PIN protein family, was the first protein to be characterized as an intracellular auxin transporter. We demonstrated that PIN8, the closest member of the PIN family to PIN5, represents another ER-residing auxin transporter. PIN8 is specifically expressed in the male gametophyte and is located in the ER. By combining genetic, physiological, cellular and biochemical data we demonstrated a role for PIN8 in intracellular auxin homeostasis. Although our investigation shed light on intracellular auxin transport in pollen, the physiological function of PIN8 still remains to be elucidated. Here we discuss our data taking in consideration other recent findings.

Sugarbeet (Beta vulgaris L.): shoot regeneration from callus and callus protoplasts.

The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.