Showing papers by "Klaus Palme published in 2013"

Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex.

Abstract: 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.

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Abstract: Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.

Plastid-Localized Glutathione Reductase2–Regulated Glutathione Redox Status Is Essential for Arabidopsis Root Apical Meristem Maintenance

Abstract: Glutathione is involved in thiol redox signaling and acts as a major redox buffer against reactive oxygen species, helping to maintain a reducing environment in vivo. Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH). The Arabidopsis thaliana genome encodes two GRs: GR1 and GR2. Whereas the cytosolic/peroxisomal GR1 is not crucial for plant development, we show here that the plastid-localized GR2 is essential for root growth and root apical meristem (RAM) maintenance. We identify a GR2 mutant, miao, that displays strong inhibition of root growth and severe defects in the RAM, with GR activity being reduced to ∼50%. miao accumulates high levels of GSSG and exhibits increased glutathione oxidation. The exogenous application of GSH or the thiol-reducing agent DTT can rescue the root phenotype of miao, demonstrating that the RAM defects in miao are triggered by glutathione oxidation. Our in silico analysis of public microarray data shows that auxin and glutathione redox signaling generally act independently at the transcriptional level. We propose that glutathione redox status is essential for RAM maintenance through both auxin/PLETHORA (PLT)-dependent and auxin/PLT-independent redox signaling pathways.

Inactivation of plasma membrane-localized CDPK-RELATED KINASE5 decelerates PIN2 exocytosis and root gravitropic response in Arabidopsis.

Abstract: CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

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

Abstract: 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.

ERECTA Family Genes Regulate Auxin Transport in the Shoot Apical Meristem and Forming Leaf Primordia

Abstract: Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes.

Abstract: Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

Variational attenuation correction in two-view confocal microscopy

Abstract: Background Absorption and refraction induced signal attenuation can seriously hinder the extraction of quantitative information from confocal microscopic data. This signal attenuation can be estimated and corrected by algorithms that use physical image formation models. Especially in thick heterogeneous samples, current single view based models are unable to solve the underdetermined problem of estimating the attenuation-free intensities.

Discriminative Detection and Alignment in Volumetric Data

Abstract: In this paper, we aim for detection and segmentation of Arabidopsis thaliana cells in volumetric image data. To this end, we cluster the training samples by their size and aspect ratio and learn a detector and a shape model for each cluster. While the detector yields good cell hypotheses, additionally aligning the shape model to the image allows to better localize the detections and to reconstruct the cells in case of low quality input data. We show that due to the more accurate localization, the alignment also improves the detection performance.

Joint 3D cell segmentation and classification in the Arabidopsis root using energy minimization and shape priors

Abstract: This paper presents a discrete energy minimization approach to integrate different prior knowledge and image cues for simultaneous cell segmentation and classification. When there are multiple types of cells to segment, the segmentation of cells and the classification of the cell types are dependent on each other. The presented approach selects the optimal segmentations from hypotheses and infers the cell types in the same process. The approach is applied to the volumetric data of Arabidopsis roots.

Variational attenuation correction of two-view confocal microscopic recordings

Abstract: We present an approach to recover attenuation-free intensities of a thick sample, that is imaged by a standard confocal microscope from two views (top and bottom). A variational approach simultaneously estimates the local signal attenuation and the real attenuation-free intensity at each position. Compared to earlier work we introduce a refined image formation model, that models photo-bleaching and photon noise using Poisson image statistics. We examine the effects of different regularization methods on the absorption field (Tikhonov-Miller, Total Variation, and sparsity) and the benefit of a constrained optimization in comparison to an orthogonal subspace projection. We quantify the efficacy of the approach on synthetically generated samples, and show its general applicability on two real biological applications, namely the recordings of zebrafish embryos and Arabidopsis thaliana root tips.

Root systems analysis branches out

Abstract: Mol Syst Biol. 9: 698 Land plants secure a constant supply of water and nutrients by developing a deep and densely branched root system. Nutrients are often distributed unevenly in the soil; formation of lateral roots must therefore be responsive to environmental conditions. Over time, lateral root initiation has become an accessible model system for studying the basic regulatory mechanisms that control postembryonic organ development. While there has been much interest in the phenomenology of root branching, it is only recently that the underlying molecular processes have started to be understood (Malamy, 2005). The recent article by Bennett and colleagues (Peret et al , 2013) presents an exciting example of how an integrated experimental and mathematical approach can elucidate the signaling pathways that drive lateral root emergence. The authors chose their model plant wisely: Arabidopsis thaliana has a relatively simple root anatomy when compared to other plants such as grasses. Arabidopsis roots are composed of single concentric layers of epidermal, cortical, endodermal and pericycle cells, which surround a double‐stranded vascular cylinder (Figure 1). Lateral roots originate from a …