Azelaic acid can efficiently compete for the auxin binding site TIR1, altering auxin polar transport, gravitropic response, and root growth and architecture in Arabidopsisthaliana roots.

The present study investigates the phytotoxic potential of azelaic acid (AZA) on Arabidopsis thaliana roots. Effects on root morphology, anatomy, auxin content and transport, gravitropic response and molecular docking were analysed. AZA inhibited root growth, stimulated lateral and adventitious roots, and altered the root apical meristem by reducing meristem cell number, length and width. The treatment also slowed down the roots' gravitropic response, likely due to a reduction in statoliths, starch-rich organelles involved in gravity perception. In addition, auxin content, transport and distribution, together with PIN proteins' expression and localisation were altered after AZA treatment, inducing a reduction in auxin transport and its distribution into the meristematic zone. Computational simulations showed that AZA has a high affinity for the auxin receptor TIR1, competing with auxin for the binding site. The AZA binding with TIR1 could interfere with the normal functioning of the TIR1/AFB complex, disrupting the ubiquitin E3 ligase complex and leading to alterations in the response of the plant, which could perceive AZA as an exogenous auxin. Our results suggest that AZA mode of action could involve the modulation of auxin-related processes in Arabidopsis roots. Understanding such mechanisms could lead to find environmentally friendly alternatives to synthetic herbicides.

Nitric oxide, gravity response, and a unified schematic of plant signaling.

Plant signaling components are often involved in numerous processes. Calcium, reactive oxygen species, and other signaling molecules are essential to normal biotic and abiotic responses. Yet, the summation of these components is integrated to produce a specific response despite their involvement in a myriad of response cascades. In the response to gravity, the role of many of these individual components has been studied, but a specific sequence of signals has not yet been assembled into a cohesive schematic of gravity response signaling. Herein, we provide a review of existing knowledge of gravity response and differential protein and gene regulation induced by the absence of gravity stimulus aboard the International Space Station and propose an integrated theoretical schematic of gravity response incorporating that information. Recent developments in the role of nitric oxide in gravity signaling provided some of the final contextual pillars for the assembly of the model, where nitric oxide and the role of cysteine S-nitrosation may be central to the gravity response. The proposed schematic accounts for the known responses to reorientation with respect to gravity in roots-the most well studied gravitropic plant tissue-and is supported by the extensive evolutionary conservation of regulatory amino acids within protein components of the signaling schematic. The identification of a role of nitric oxide in regulating the TIR1 auxin receptor is indicative of the broader relevance of the schematic in studying a multitude of environmental and stress responses. Finally, there are several experimental approaches that are highlighted as essential to the further study and validation of this schematic.

AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root.

The membrane potential reflects the difference between cytoplasmic and apoplastic electrical potentials and is essential for cellular operation. The application of the phytohormone auxin (3-indoleacetic acid (IAA)) causes instantaneous membrane depolarization in various cell types1-6, making depolarization a hallmark of IAA-induced rapid responses. In root hairs, depolarization requires functional IAA transport and TIR1-AFB signalling5, but its physiological importance is not understood. Specifically in roots, auxin triggers rapid growth inhibition7-9 (RGI), a process required for gravitropic bending. RGI is initiated by the TIR1-AFB co-receptors, with the AFB1 paralogue playing a crucial role10,11. The nature of the underlying rapid signalling is unknown, as well as the molecular machinery executing it. Even though the growth and depolarization responses to auxin show remarkable similarities, the importance of membrane depolarization for root growth inhibition and gravitropism is unclear. Here, by combining the DISBAC2(3) voltage sensor with microfluidics and vertical-stage microscopy, we show that rapid auxin-induced membrane depolarization tightly correlates with RGI. Rapid depolarization and RGI require the AFB1 auxin co-receptor. Finally, AFB1 is essential for the rapid formation of the membrane depolarization gradient across the gravistimulated root. These results clarify the role of AFB1 as the central receptor for rapid auxin responses.

OsARF11 Promotes Growth, Meristem, Seed, and Vein Formation during Rice Plant Development.

The plant hormone auxin acts as a mediator providing positional instructions in a range of developmental processes. Studies in Arabidopsis thaliana L. show that auxin acts in large part via activation of Auxin Response Factors (ARFs) that in turn regulate the expression of downstream genes. The rice (Oryza sativa L.) gene OsARF11 is of interest because of its expression in developing rice organs and its high sequence similarity with MONOPTEROS/ARF5, a gene with prominent roles in A. thaliana development. We have assessed the phenotype of homozygous insertion mutants in the OsARF11 gene and found that in relation to wildtype, osarf11 seedlings produced fewer and shorter roots as well as shorter and less wide leaves. Leaves developed fewer veins and larger areoles. Mature osarf11 plants had a reduced root system, fewer branches per panicle, fewer grains per panicle and fewer filled seeds. Mutants had a reduced sensitivity to auxin-mediated callus formation and inhibition of root elongation, and phenylboronic acid (PBA)-mediated inhibition of vein formation. Taken together, our results implicate OsARF11 in auxin-mediated growth of multiple organs and leaf veins. OsARF11 also appears to play a central role in the formation of lateral root, panicle branch, and grain meristems.

Latrunculin B facilitates gravitropic curvature of Arabidopsis root by inhibiting cell elongation, especially the cells in the lower flanks of the transition and elongation zones.

Gravitropism plays a critical role in the growth and development of plants. Previous reports proposed that the disruption of the actin cytoskeleton resulted in enhanced gravitropism; however, the mechanism underlying these phenomena is still unclear. In the present study, real-time observation on the effect of Latrunculin B (Lat B), a depolymerizing agent of microfilament cytoskeleton, on gravitropism of the primary root of Arabidopsis was undertaken using a vertical stage microscope. The results indicated that Lat B treatment prevented the growth of root, and the growth rates of upper and lower flanks of the horizontally placed root were asymmetrically inhibited. The growth of the lower flank was influenced by Lat B more seriously, resulting in an increased differential growth rate between the upper and lower flanks of the root. Further analysis indicated that Lat B affected cell growth mainly in the transition and elongation zones. Briefly, the current data revealed that Lat B treatment inhibited cell elongation, especially the cells in the lower flanks of the transition and elongation zones, which finally manifested as the facilitation of gravitropic curvature of the primary root.

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance.

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.

Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter.

Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.

Involvement of BIG5 and BIG3 in BRI1 Trafficking Reveals Diverse Functions of BIG-subfamily ARF-GEFs in Plant Growth and Gravitropism.

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking.

Pinstatic Acid Promotes Auxin Transport by Inhibiting PIN Internalization.

Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.

Effects of exogenous 24-epibrassinolide and brassinazole on negative gravitropism and tension wood formation in hybrid poplar (Populus deltoids × Populus nigra).

MAIN CONCLUSION: Exogenous 24-epibrassinolide (BL) and brassinazole (BRZ) have regulatory roles in G-fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during tension wood formation in hybrid poplar. Brassinosteroids (BRs) play important roles in regulating gravitropism and vasculature development. Here, we report the effect of brassinosteroids on negative gravitropism and G-fiber cell wall development of the stem in woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. Our findings provide potential insights into the mechanism by which BRs regulate G-fiber cell wall development to accomplish negative gravitropism in TW formation.

Omeprazole Enhances Mechanical Stress-Induced Root Growth Reduction in Arabidopsis thaliana.

Mechanical sensing is one of the most fundamental processes for sessile plants to survive and grow. The response is known to involve calcium elevation in the cell. Arabidopsis seedlings grown horizontally on agar plates covered with a dialysis membrane show a 2-fold reduction in root growth compared with those grown vertically, a response to mechanical stress generated due to gravitropism of the root. To understand the molecular mechanism of how plant roots sense and respond to mechanical stimuli, we screened chemical libraries for compounds that affect the horizontal root growth in this experimental system and found that, while having no effect on root gravitropism, omeprazole known as a proton pump inhibitor significantly enhanced the mechanical stress-induced root growth reduction especially in lower pH media. In contrast, omeprazole reversed neither the alleviation of the mechanical stress-induced growth reduction caused by calcium depletion nor the insensitivity to the mechanical stress in the ethylene signaling mutant ein2. Together with the finding that omeprazole increased expression of touch-induced genes and ETHYLENE RESPONSE FACTOR1, our results suggest that the target of omeprazole mediates ethylene signaling in the root growth response to mechanical stress.

The chemical NJ15 affects hypocotyl elongation and shoot gravitropism via cutin polymerization.

We previously found a chemical, designated as NJ15, which inhibited both auxin and brassinosteroid responses in dark-grown Arabidopsis. To study its mode of action, we performed a phenotypic screening of NJ15-low-sensitive lines among mutant pools of Arabidopsis. One line (f127) showed clear NJ15-low-sensitivity in terms of hypocotyl elongation and shoot gravitropism. After further testing, it was determined that DCR, an enzyme involved in cutin polymerization, had lost its function in the mutant, which caused its low sensitivity to NJ15. Fatty acids are the base materials for polymers such as cutin and cuticular wax. We confirmed that NJ15 affects fatty acid biosynthesis, and that it does differently from cafenstrole, a known inhibitor of cuticular wax formation. Based on these results, we propose that the target of NJ15 is likely located within the cutin polymer formation pathway. ABBREVIATIONS: Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid.

Exogenous hydrogen peroxide inhibits primary root gravitropism by regulating auxin distribution during Arabidopsis seed germination.

Hydrogen peroxide (H2O2) is the key factor in many physiological and metabolic processes in plants. During seed germination, exogenous H2O2 application influences gravitropism and induces curvature of the primary root in grass pea and pea seedlings. However, it remains unclear whether and how this happens in the model plant Arabidopsis thaliana. In the present study, the effect of exogenous H2O2 on the gravitropic response of primary roots during Arabidopsis seed germination was studied using histology and molecular biology approaches. Appropriate H2O2 treatment not only restrained primary root growth, but also disrupted gravitropism and induced root curvature. Histological staining and molecular analysis demonstrated that exogenous H2O2 correlated with lack of starch-dense amyloplasts in root tip columella cells, which ultimately results in the lack of gravisensing. Detection of calcium ion (Ca2+) by a fluorescent probe showed that Ca2+ distribution changed and intracellular Ca2+ concentration increased in H2O2-treated primary root, which was consistent with alterations in auxin distribution and concentration triggered by H2O2 treatment. Furthermore, the normally polar localization of Pin-formed 1 (PIN1) and PIN2 became uniformly distributed on root tip cell membranes after treatment with H2O2. This leads to speculation that the IAA signaling pathway was affected by exogenous H2O2, causing asymmetrical distribution of IAA on both sides of the primary root, which would influence the gravitropic response.

The effect of auxin (indole-3-acetic acid) on the growth rate and tropism of the sporangiophore of Phycomyces blakesleeanus and identification of auxin-related genes.

The roles of fungal auxins in the regulation of elongation growth, photo-, and gravitropism are completely unknown. We analyzed the effects of exogenous IAA (indole-3-acetic acid), various synthetic auxins including 1-NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the auxin transport inhibitor NPA (N-1-naphtylphtalamic acid) on the growth rate and bending of the unicellular sporangiophore of the zygomycete fungus, Phycomyces blakesleeanus. Sporangiophores that were submerged in an aqueous buffer responded to IAA with a sustained enhancement of the growth rate, while 1-NAA, 2,4-D, and NPA elicited an inhibition. In contrast, sporangiophores kept in air responded to IAA with a 20 to 40% decrease of the growth rate, while 1-NAA and NPA elicited an enhancement. The unilateral and local application of IAA in the growing zone of the sporangiophore elicited in 30 min a moderate negative tropic bending in wild type C2 and mutant C148madC, which was, however, partially masked by a concomitant avoidance response caused by the aqueous buffer. Auxin transport-related genes ubiquitous in plants were found in a BLAST search of the Phycomyces genome. They included members of the AUX1 (auxin influx carrier protein 1), PILS (PIN-LIKES, auxin transport facilitator protein), and ABCB (plant ATP-binding cassette transporter B) families while members of the PIN family were absent. Our observations imply that IAA represents an intrinsic element of the sensory transduction of Phycomyces and that its mode of action must very likely differ in several respects from that operating in plants.

Toxicity of silver nanoparticles to Arabidopsis: Inhibition of root gravitropism by interfering with auxin pathway.

Impacts of polyvinylpyrrolidine-coated silver nanoparticles (AgNPs) on root gravitropism in Arabidopsis thaliana were investigated at the physiological, cellular, and molecular levels. Our results showed that AgNPs were taken up by the root and primarily localized at the cell wall and intercellular spaces. Root gravitropism was inhibited by exposure to AgNPs, and the inhibition in root gravitropism caused by exposure to AgNPs exhibited a dose-response relationship. Auxin accumulation was reduced in the root tips because of exposure to AgNPs. However, increased indole-3-acetic acid level could not rescue the inhibition of root gravitropism. Real-time polymerase chain reaction showed significant downregulation of expression of auxin receptor-related genes, which is the TIR1/AFB family of F-box proteins including AFB1, AFB2, AFB3, AFB5, and TIR1. Therefore, the present study suggests that AgNPs have toxicity to the model plant A. thaliana as shown by inhibition of root gravitropism along with a reduction in auxin accumulation and expression of auxin receptors. Environ Toxicol Chem 2017;36:2773-2780. © 2017 SETAC.

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls.

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.

EHB1 and AGD12, two calcium-dependent proteins affect gravitropism antagonistically in Arabidopsis thaliana.

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca2+-activated states.