An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endo-membrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development

A network of stress-related genes regulates hypocotyl elongation downstream of selective auxin perception

The plant hormone auxin, a master coordinator of development, regulates hypocotyl elongation during seedling growth. We previously identified the synthetic molecule RubNeddin 1 (RN1), which induces degradation of the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors INDOLE-3-ACETIC ACID-INDUCIBLE3 (IAA3) and IAA7 in planta and strongly promotes hypocotyl elongation. In the present study, we show that despite the structural similarity of RN1 to the synthetic auxin 2,4-dichlorophenoxyacetic-acid (2,4-D), direct treatments with these compounds in Arabidopsis (Arabidopsis thaliana) result in distinct effects, possibly due to enhanced uptake of RN1 and low-level, chronic release of 2,4-D from RN1 in planta. We confirm RN1-induced hypocotyl elongation occurs via specific TRANSPORT INHIBITOR RESISTANT1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) receptor-mediated auxin signaling involving TIR1, AFB2, and AFB5. Using a transcriptome profiling strategy and candidate gene approach, we identify the genes ZINC FINGER OF ARABIDOPSIS THALIANA10 (ZAT10), ARABIDOPSIS TOXICOS EN LEVADURA31 (ATL31), and WRKY DNA-BINDING PROTEIN33 (WRKY33) as being rapidly upregulated by RN1, despite being downregulated by 2,4-D treatment. RN1-induced expression of these genes also occurs via TIR1/AFB-mediated auxin signaling. Our results suggest both hypocotyl elongation and transcription of these genes are induced by RN1 via the promoted degradation of the AUX/IAA transcriptional repressor IAA7. Moreover, these three genes, which are known to be stress-related, act in an inter-dependent transcriptional regulatory network controlling hypocotyl elongation. Together, our results suggest ZAT10, ATL31, and WRKY33 take part in a common gene network regulating hypocotyl elongation in Arabidopsis downstream of a selective auxin perception module likely involving TIR1, AFB2, and AFB5 and inducing the degradation of IAA7

An AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition

Abstract Differentiation of xylem elements involves cell expansion, secondary cell wall deposition and programmed cell death. Transitions between these phases require strict spatiotemporal control. The function of Populus ERF139 (Potri.013G101100) in xylem differentiation was characterized in transgenic overexpression and dominant repressor lines of ERF139 in hybrid aspen (Populus tremula x tremuloides). Xylem properties, secondary cell wall (SCW) chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, FT-IR, pyrolysis-GC/MS, wet chemistry methods and RNA sequencing. Opposite phenotypes were observed in the secondary xylem vessel sizes and SCW chemistry in the two different types of transgenic trees, supporting the function of ERF139 in suppressing the radial expansion of vessel elements and stimulating accumulation of guaiacyl-type lignin and possibly also xylan. Comparative transcriptomics identified genes related to SCW biosynthesis (LAC5, LBD15, MYB86) and salt and drought stress responsive genes (ANAC002, ABA1) as potential direct targets of ERF139. The phenotypes of the transgenic trees and the stem expression profiles of ERF139 potential target genes support the role of ERF139 as a transcriptional regulator of xylem cell expansion and SCW formation, possibly in response to osmotic changes of the cells. This article is protected by copyright. All rights reserved.Peer reviewe

New PEO-IAA-Inspired Anti-Auxins: Synthesis, Biological Activity, and Possible Application in Hemp (Cannabis Sativa L.) Micropropagation

Auxins play an important role in plant physiology and are involved in numerous aspects of plant development, such as cell division, elongation and differentiation, fruit development, and phototropic response. In addition, through their antagonistic interaction with cytokinins, auxins play a key role in the regulation of root growth and apical dominance. Thanks to this capacity to determine plant architecture, natural and synthetic auxins have been successfully employed to obtain more economically advantageous plants. The crosstalk between auxins and cytokinins determines plant development and thus is of particular importance in the field of plant micropropagation, where the ratios between these two phytohormones need to be tightly controlled to achieve proper rooting and shoot generation. Previously reported anti-auxin PEO-IAA, which blocks auxin signalling through binding to TIR1 receptor and inhibiting the expression of auxin-responsive genes, has been successfully used to facilitate hemp micropropagation. Herein, we report a set of new PEO-IAA-inspired anti-auxins capable of antagonizing auxin responses in vivo. The capacity of these compounds to bind to the TIR1 receptor was confirmed in vitro by SPR analysis. Using DESI-MSI analysis, we evaluated the uptake and distribution of the compounds at the whole plant level. Finally, we characterized the effect of the compounds on the organogenesis of hemp explants, where they showed to be able to improve beneficial morphological traits, such as the balanced growth of all the produced shoots and enhanced bud proliferation

Mechanochemical Polarization of Contiguous Cell Walls Shapes Plant Pavement Cells.

The epidermis of aerial plant organs is thought to be limiting for growth, because it acts as a continuous load-bearing layer, resisting tension. Leaf epidermis contains jigsaw puzzle piece-shaped pavement cells whose shape has been proposed to be a result of subcellular variations in expansion rate that induce local buckling events. Paradoxically, such local compressive buckling should not occur given the tensile stresses across the epidermis. Using computational modeling, we show that the simplest scenario to explain pavement cell shapes within an epidermis under tension must involve mechanical wall heterogeneities across and along the anticlinal pavement cell walls between adjacent cells. Combining genetics, atomic force microscopy, and immunolabeling, we demonstrate that contiguous cell walls indeed exhibit hybrid mechanochemical properties. Such biochemical wall heterogeneities precede wall bending. Altogether, this provides a possible mechanism for the generation of complex plant cell shapes

Selective auxin agonists induce specific AUX/IAA protein degradation to modulate plant development.

Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses

Chemical biology and digital image processing to unravel complex molecular mechanisms in Arabidopsis

The sessile life of plant directed their evolution toward multiple adaptive strategies. Rapid protein turn over has been described to be a key regulatory mechanism for plant adaptation. Ubiquitin-modified proteins are targeted for degradation by the 26S-proteasome. A class of ubiquitin-ligases, the Cullin Ring Ligases (CRLs) have been shown to be involved in most Arabidopsis developmental processes. CRLs are stabilized by the covalent binding of the small peptide RELATED TO UBIQUITIN (RUB). The CRLs modification is required for the activity of plant hormones as exemplified by mutants deficient in the RUB-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). The perception of auxin results in the ubiquitin-mediated degradation of the AUXIN/INDOL-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Aux/IAA proteins control the auxin-mediated transcriptional response. In this work, a forward chemical genomic strategy has been used to identify small synthetic molecules affecting plant development. We used the resistance of the axr1-30 mutants in order to select compounds requiring RUB activation. Among the molecules isolated to alter specific plant developmental processes, three Developmental Regulators (DRs) have been shown to directly interfere with the degradation of the Aux/IAA proteins promoting a rapid induction of specific auxin-related transcriptional responses. Furthermore, we used the molecule DR4, abolishing specifically apical hook formation, to investigate the functional selectivity of auxin perception during apical hook development. A forward genetic screen has been performed to isolate dr4-resistant mutants. Several viable mutants were isolated with different sensitivity to auxin but all resistant to DR4. The isolation of mutants preferentially resistant to the differential growth defect induced by DR4 demonstrates the potential to determine the molecular process mediating the developmental features induced by the selective agonists of auxin. Since the first digital image 60 years ago, imaging techniques are constantly evolving generating more and more digital images. The conversion of images into biologically relevant quantitative data is an essential process to overcome and understand biological variability. In this work, we describe two digital images processing approaches which have been used to semi-automatically describe intracellular structure density and colocalization; the complex shape of the Arabidopsis pavement cells