Showing papers by "Klaus Palme published in 2016"

A detailed expression map of the PIN1 auxin transporter in Arabidopsis thaliana root.

Abstract: Theauxin efflux carrier PIN1 is a key mediator of polar auxin transport in developing plant tissues. This is why factors that are supposed to be involved in auxin distribution are frequently tested in the regulation of PIN1 expression. As a result, diverse aspects of PIN1 expression are dispersed across dozens of papers entirely devoted to other specific topics related to the auxin pathway. Integration of these puzzle pieces about PIN1 expression revealed that, along with a recurring pattern, some features of PIN1 expression varied from article to article. To determine if this uncertainty is related to the specific foci of articles or has a basis in the variability of PIN1 gene activity, we performed a comprehensive 3D analysis of PIN1 expression patterns in Arabidopsis thaliana roots. We provide here a detailed map of PIN1 expression in the primary root, in the lateral root primordia and at the root-shoot junction. The variability in PIN1 expression pattern observed in individual roots may occur due to differences in auxin distribution between plants. To simulate this effect, we analysed PIN1 expression in the roots from wild type seedlings treated with different IAA concentrations and pin mutants. Most changes in PIN1 expression after exogenous IAA treatment and in pin mutants were also recorded in wild type but with lower frequency and intensity. Comparative studies of exogenous auxin effects on PIN1pro:GUS and PIN1pro:PIN1-GFP plants indicated that a positive auxin effect is explicit at the level of PIN1 promoter activity, whereas the inhibitory effect relates to post-transcriptional regulation. Our results suggest that the PIN1 expression pattern in the root meristem accurately reflects changes in auxin content. This explains the variability of PIN1 expression in the individual roots and makes PIN1 a good marker for studying root meristem activity.

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

Abstract: 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.

Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

Abstract: Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

2-D Clinostat for Simulated Microgravity Experiments with Arabidopsis Seedlings

Abstract: Ground-based simulators of microgravity such as fast rotating 2-D clinostats are valuable tools to study gravity related processes. We describe here a versatile g-value-adjustable 2-D clinostat that is suitable for plant analysis. To avoid seedling adaptation to 1 g after clinorotation, we designed chambers that allow rapid fixation. A detailed protocol for fixation, RNA isolation and the analysis of selected genes is described. Using this clinostat we show that mRNA levels of LONG HYPOCOTYL 5 (HY5), MIZU-KUSSEI 1 (MIZ1) and microRNA MIR163 are down-regulated in 5-day-old Arabidopsis thaliana roots after 3 min and 6 min of clinorotation using a maximal reduced g-force of 0.02 g, hence demonstrating that this 2-D clinostat enables the characterization of early transcriptomic events during root response to microgravity. We further show that this 2-D clinostat is able to compensate the action of gravitational force as both gravitropic-dependent statolith sedimentation and subsequent auxin redistribution (monitoring D R5 r e v ::G F P reporter) are abolished when plants are clinorotated. Our results demonstrate that 2-D clinostats equipped with interchangeable growth chambers and tunable rotation velocity are suitable for studying how plants perceive and respond to simulated microgravity.

Novel small molecule modulators of plant growth and development identified by high-content screening with plant pollen

Abstract: Small synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development. We developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %. In this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.

Inhibition of Cell Expansion by Rapid ABP1-Mediated Auxin Effect on Microtubules? A Critical Comment.

Abstract: How do cortical microtubules shape plant cells? This has been an important question ever since the microtubular cytoskeleton was found to orientate the deposition of cellulose microfibrils in the primary cell wall and control long-term anisotropic cell expansion under isotropic turgor pressure ([

Erratum to: 2-D Clinostat for Simulated Microgravity Experiments with Arabidopsis Seedlings

Abstract: 6 School of Biological Science and Technology, University of Jinan, 336, West Road of Nan Xinzhuang, Jinan 250022, China Wang et al: 2-D Clinostat for Simulated Microgravity Experiments with Arabidopsis Seedlings Microgravity Sci Technol 28, 59–66 (2016) doi:101007/s12217-015-94781 was published in an earlier issue instead of being included in this “Topical issue on Ground-Based Facilities”

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family (small guanine nucleotide binding protein/ras gene superfamily)

Abstract: We have isolated, cloned, and characterized two cDNAs from Zea mays (L), denoted yptml and yptm2, encoding proteins related to the ypt protein family Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptml and 74% for yptm2, respec- tively, whereas similarities with p21 ras and other ras-related proteins are <40% Most amino acid residues showing identity are clustered in the GTP/GDP binding domain In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs The gene for yptml is expressed at very low levels in maize coleoptiles and tissue culture cells The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers The structural similarity of the genes identified sug- gests that they could be involved in the control of secretory processes