Alexander Dovzhenko

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.

Auxin as a Model for the Integration of Hormonal Signal Processing and Transduction

The regulation of plant growth responds to many stimuli. These responses allow environmental adaptation, thereby increasing fitness. In many cases, the relay of information about a plants environment is through plant hormones. These messengers integrate environmental information into developmental pathways to determine plant shape. This review will use, as an example, auxin in the root of Arabidopsis thaliana to illustrate the complex nature of hormonal signal processing and transduction. It will then make the case that the application of a systems-biology approach is necessary, if the relationship between a plants environment and its growth/developmental responses is to be properly understood.

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.

Auxin transport and gravitational research: perspectives

Summary.Gravity is a fundamental factor which affects all living organisms. Plant development is well adapted to gravity by directing roots downward and shoots upwards. For more than a century, plant biologists have been fascinated to describe the molecular mechanisms underlying the gravitropic response of plants. Important progress towards signal perception, transduction, and response has been made, but new tools are beginning to uncover the regulatory networks for gravitropic control. We summarise recent progress in study of gravitropism and discuss strategies to identify the molecular basis of the gravity response in Arabidopsis thaliana. This will put us on a road towards the molecular systems biology of the Arabidopsis gravitropic response.

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 roots 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.

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.

Fast scalar and vectorial grayscale based invariant features for 3d cell nuclei localization and classification

Since biology and medicine apply increasingly fast volumetric imaging techniques and aim at extracting quantitative data from these images, the need for efficient image analysis techniques like detection and classification of 3D structures is obvious. A common approach is to extract local features, e.g. group integration has been used to gain invariance against rotation and translation. We extend these group integration features by including vectorial information and spherical harmonics descriptors. From our vectorial invariants we derive a very robust detector for spherical structures in low-quality images and show that it can be computed very fast. We apply these new invariants to 3D confocal laser-scanning microscope images of the Arabidopsis root tip and extract position and type of the cell nuclei. Then it is possible to build a biologically relevant, architectural model of the root tip.

Analysis of gene expression during parabolic flights reveals distinct early gravity responses in Arabidopsis roots

Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root responses to gravity, being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana wild type, and pin2 and pin3 mutants under parabolic flight conditions and to correlate these changes to auxin distribution. Parabolic flights allow comparison of transient 1-g, hypergravity and microgravity effects in living organisms in parallel. We found common and mutation-related genes differentially expressed in response to transient microgravity phases. Gene ontology analysis of common genes revealed lipid metabolism, response to stress factors and light categories as primarily involved in response to transient microgravity phases, suggesting that fundamental reorganisation of metabolic pathways functions upstream of a further signal mediating hormonal network. Gene expression changes in roots lacking the columella-located PIN3 were stronger than in those deprived of the epidermis and cortex cell-specific PIN2. Moreover, repetitive exposure to microgravity/hypergravity and gravity/hypergravity flight phases induced an up-regulation of auxin responsive genes in wild type and pin2 roots, but not in pin3 roots, suggesting a critical function of PIN3 in mediating auxin fluxes in response to transient microgravity phases. Our study provides important insights towards understanding signal transduction processes in transient microgravity conditions by combining for the first time the parabolic flight platform with the transcriptome analysis of different genetic mutants in the model plant, Arabidopsis.

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.

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.