Characterization of OsPIN2 Mutants Reveal Novel Roles for Reactive Oxygen Species in Modulating Not Only Root Gravitropism but Also Hypoxia Tolerance in Rice Seedlings.

Tolerance to submergence-induced hypoxia is an important agronomic trait especially for crops in lowland and flooding-affected areas. Although rice (Oryza sativa) is considered a flood-tolerant crop, only limited cultivars display strong tolerance to prolonged submergence and/or hypoxic stress. Therefore, characterization of hypoxic resistant genes and/or germplasms have important theoretical and practical significance for rice breeding and sustained improvements. Previous investigations have demonstrated that loss-of-function of OsPIN2, a gene encoding an auxin efflux transporter, results in the loss of root gravitropism due to disrupted auxin transport in the root tip. In this study, we revealed a novel connection between OsPIN2 and reactive oxygen species (ROS) in modulating root gravitropism and hypoxia tolerance in rice. It is shown that the OsPIN2 mutant had decreased accumulation of ROS in root tip, due to the downregulation of glycolate oxidase encoding gene OsGOX6, one of the main H2O2 sources. The morphological defects of root including waved rooting and agravitropism in OsPIN2 mutant may be rescued partly by exogenous application of H2O2. The OsPIN2 mutant exhibited increased resistance to ROS toxicity in roots due to treatment with H2O2. Furthermore, it is shown that the OsPIN2 mutant had increased tolerance to hypoxic stress accompanied by lower ROS accumulation in roots, because the hypoxia stress led to over production of ROS in the roots of the wild type but not in that of OsPIN2 mutant. Accordingly, the anoxic resistance-related gene SUB1B showed differential expression in the root of the WT and OsPIN2 mutant in response to hypoxic conditions. Notably, compared with the wild type, the OsPIN2 mutant displayed a different pattern of auxin distribution in the root under hypoxia stress. It was shown that hypoxia stress caused a significant increase in auxin distribution in the root tip of the WT but not in that of the war1 mutant. In summary, these results suggested that OsPIN2 may play a role in regulating ROS accumulation probably via mediating auxin transport and distribution in the root tip, affecting root gravitropism and hypoxic tolerance in rice seedlings. These findings may contribute to the genetic improvement and identification of potential hypoxic tolerant lines in rice.

The RGI1-BAK1 module acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs), a group of leucine-rich repeat receptor kinases, promote primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade and control root gravitropism in Arabidopsis. Genetic analyses and in vitro binding assays have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 recognize RGF1 peptides. However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in the regulation of primary root meristem activity. In the present study, we analyzed root meristem growth of the rgi1, rgi2, and rgi3 single mutants in response to RGF1 treatment and observed a significantly decreased sensitivity in meristem growth of rgi1 and complete insensitivity in rgi1 rgi2 rgi3 triple mutant compared with the wild type but not in the rgi1 and rgi2 single mutants. We also observed that both root gravitropism and meristem growth in the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (bak1) single mutant were insensitive to RGF1 peptide treatment, whereas other serk mutants, such as serk1, serk2, and serk4, were fully sensitive to RGF1 peptide like the wild type. These mutant analyses suggest that RGI1-BAK1 pair acts as the main receptor-coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

Short-term exposition to acute cadmium toxicity induces the loss of root gravitropic stimuli perception through PIN2-mediated auxin redistribution in Arabidopsis thaliana (L.) Heynh.

Cadmium (Cd), one of the most widespread and water-soluble polluting heavy metals, has been widely studied on plants, even if the mechanisms underlying its phytotoxicity remain elusive. Indeed, most experiments are performed using extensive exposure time to the toxicants, not observing the primary targets affected. The present work studied Cd effects on Arabidopsis thaliana (L.) Heynh's root apical meristem (RAM) exposed for short periods (24 h and 48 h) to acute phytotoxic concentrations (100 and 150 µM). The effects were studied through integrated morpho-histological, molecular, pharmacological and metabolomic analyses, highlighting that Cd inhibited primary root elongation by affecting the meristem zone via altering cell expansion. Moreover, Cd altered Auxin accumulation in RAM and affected PINs polar transporters, particularly PIN2. In addition, we observed that high Cd concentration induced accumulation of reactive oxygen species (ROS) in roots, which resulted in an altered organization of cortical microtubules and the starch and sucrose metabolism, altering the statolith formation and, consequently, the gravitropic root response. Our results demonstrated that short Cd exposition (24 h) affected cell expansion preferentially, altering auxin distribution and inducing ROS accumulation, which resulted in an alteration of gravitropic response and microtubules orientation pattern.

Local auxin biosynthesis regulates brace root angle and lodging resistance in maize.

Root lodging poses a major threat to maize production, resulting in reduced grain yield and quality, and increased harvest costs. Here, we combined expressional, genetic, and cytological studies to demonstrate a role of ZmYUC2 and ZmYUC4 in regulating gravitropic response of the brace root and lodging resistance in maize. We show that both ZmYUC2 and ZmYUC4 are preferentially expressed in root tips with partially overlapping expression patterns, and the protein products of ZmYUC2 and ZmYUC4 are localized in the cytoplasm and endoplasmic reticulum, respectively. The Zmyuc4 single mutant and Zmyuc2/4 double mutant exhibit enlarged brace root angle compared with the wild-type plants, with larger brace root angle being observed in the Zmyuc2/4 double mutant. Consistently, the brace root tips of the Zmyuc4 single mutant and Zmyuc2/4 double mutant accumulate less auxin and are defective in proper reallocation of auxin in response to gravi-stimuli. Furthermore, we show that the Zmyuc4 single mutant and the Zmyuc2/4 double mutant display obviously enhanced root lodging resistance. Our combined results demonstrate that ZmYUC2- and ZmYUC4-mediated local auxin biosynthesis is required for normal gravity response of the brace roots and provide effective targets for breeding root lodging resistant maize cultivars.

Diversity of root hydrotropism among natural variants of Arabidopsis thaliana.

Root gravitropism affects root hydrotropism. The interference intensity of root gravitropism with root hydrotropism differs among plant species. However, these differences have not been well compared within a single plant species. In this study, we compared root hydrotropism in various natural variants of Arabidopsis under stationary conditions. As a result, we detected a range of root hydrotropism under stationary conditions among natural Arabidopsis variants. Comparison of root gravitropism and root hydrotropism among several Arabidopsis natural variants classified natural variants that decreased root hydrotropism into two types; namely one type that expresses root gravitropism and root hydrotropism weaker than Col-0, and the other type that expresses weaker root hydrotropism than Col-0 but expresses similar root gravitropism with Col-0. However, root hydrotropism of all examined Arabidopsis natural variants was facilitated by clinorotation. These results suggested that the interference of root gravitropism with root hydrotropism is conserved among Arabidopsis natural variants, although the intensity of root gravitropism interference with root hydrotropism differs.

ACORBA: Automated workflow to measure Arabidopsis thaliana root tip angle dynamics.

The ability of plants to sense and orient their root growth towards gravity is studied in many laboratories. It is known that manual analysis of image data is subjected to human bias. Several semi-automated tools are available for analysing images from flatbed scanners, but there is no solution to automatically measure root bending angle over time for vertical-stage microscopy images. To address these problems, we developed ACORBA, which is an automated software that can measure root bending angle over time from vertical-stage microscope and flatbed scanner images. ACORBA also has a semi-automated mode for camera or stereomicroscope images. It represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. As the software is automated, it limits human interactions and is reproducible. ACORBA will support the plant biologist community by reducing labour and increasing reproducibility of image analysis of root gravitropism.

A Multiplexed, Time-Resolved Assay of Root Gravitropic Bending on Agar Plates.

The ability of roots to orient their growth relative to the vector of gravity, root gravitropism (positive gravitropism), is observed in root systems of higher plants and is an essential part of plant growth and development. While there are various methods for quantifying root gravitropism, many methods that can efficiently measure gravitropism at a reasonable throughput do not yield temporal resolution of the process, while methods that allow for high-temporal resolution are often not suitable for an efficient measurement of multiple roots. Here, we describe a method to analyze the root gravitropism activity at an increased throughput with a fine time-resolution using Arabidopsis thaliana plants.

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis.

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O2•-) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O2•- (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed.

The Mildew Resistance Locus O 4 Interacts with CaM/CML and Is Involved in Root Gravity Response.

The plant-specific mildew resistance locus O (MLO) proteins, which contain seven transmembrane domains and a conserved calmodulin-binding domain, play important roles in many plant developmental processes. However, their mechanisms that regulate plant development remain unclear. Here, we report the functional characterization of the MLO4 protein in Arabidopsis roots. The MLO4 was identified as interacting with CML12 in a screening for the interaction between the proteins from Arabidopsis MLO and calmodulin/calmodulin-like (CaM/CML) families using yeast two hybrid (Y2H) assays. Then, the interaction between MLO4 and CML12 was further verified by Luciferase Complementation Imaging (LCI) and Bimolecular Fluorescence Complementation (BiFC) assays. Genetic analysis showed that the mlo4, cml12, and mlo4 cml12 mutants displayed similar defects in root gravity response. These results imply that the MLO4 might play an important role in root gravity response through interaction with CML12. Moreover, our results also demonstrated that the interaction between the MLO and CaM/CML families might be conservative.

Auxin and Root Gravitropism: Addressing Basic Cellular Processes by Exploiting a Defined Growth Response.

Root architecture and growth are decisive for crop performance and yield, and thus a highly topical research field in plant sciences. The root system of the model plant Arabidopsis thaliana is the ideal system to obtain insights into fundamental key parameters and molecular players involved in underlying regulatory circuits of root growth, particularly in responses to environmental stimuli. Root gravitropism, directional growth along the gravity, in particular represents a highly sensitive readout, suitable to study adjustments in polar auxin transport and to identify molecular determinants involved. This review strives to summarize and give an overview into the function of PIN-FORMED auxin transport proteins, emphasizing on their sorting and polarity control. As there already is an abundance of information, the focus lies in integrating this wealth of information on mechanisms and pathways. This overview of a highly dynamic and complex field highlights recent developments in understanding the role of auxin in higher plants. Specifically, it exemplifies, how analysis of a single, defined growth response contributes to our understanding of basic cellular processes in general.

Settling for Less: Do Statoliths Modulate Gravity Perception?

Plants orientate their growth either towards (in roots) or away from (in shoots) the Earth's gravitational field. While we are now starting to understand the molecular architecture of these gravity response pathways, the gravity receptor remains elusive. This perspective looks at the biology of statoliths and suggests it is conceivable that their immediate environment may be tuned to modulate the strength of the gravity response. It then suggests how mutant screens could use this hypothesis to identify the gravity receptor.

BES1 directly binds to the promoter of the ACC oxidase 1 gene to regulate gravitropic response in the roots of Arabidopsis thaliana.

Brassinosteroids (BRs) are known to be endogenous regulators of ethylene production, suggesting that some BR activity in plant growth and development is associated with ethylene. Here, we demonstrated that ethylene production in Arabidopsis thaliana roots is increased by BR signaling via the ethylene biosynthetic gene for ACC oxidase 1 (ACO1). Electrophoretic mobility shift and chromatin immune-precipitation assays showed that the BR transcription factor BES1 directly binds to two E-box sequences located in the intergenic region of ACO1. GUS expression using site mutations of the E-box sequences verified that ACO1 is normally expressed only when BES1 binds to the E-boxes in the putative promoter of ACO1, indicating that this binding is essential for ACO1 expression and the subsequent production of ethylene in A. thaliana roots. BR exogenously applied to A. thaliana roots enhanced the gravitropic response. Additionally, bes1-D exhibited a greater gravitropic response than did the wild-type specimens, proving that BR is a positive regulator of the gravitropic response in A. thaliana roots. The knock-down mutant aco1-1 showed a slightly lower gravitropic response than did the wild-type specimens, while bes1-D X aco1-1 exhibited a lower gravitropic response than did bes1-D. Therefore, ACO1 is a direct downstream target for BR transcription factor BES1, which controls ethylene production for gravitropism in A. thaliana roots.

LAZY3 plays a pivotal role in positive root gravitropism in Lotus japonicus.

LAZY1 family genes play important roles in both shoot and root gravitropism in plants. Here we report a Lotus japonicus mutant that displays negative gravitropic response in primary and lateral roots. Map-based cloning identified the mutant gene LAZY3 as a functional ortholog of the LAZY1 gene. Mutation of the LAZY3 gene reduced rootward polar auxin transport (PAT) in the primary root, which was also insensitive to the PAT inhibitor N-1-naphthylphthalamic acid. Moreover, immunolocalization of enhanced green fluorescent protein-tagged LAZY3 in L. japonicus exhibited polar localization of LAZY3 on the plasma membrane in root stele cells. We therefore suggest that the polar localization of LAZY3 in stele cells might be required for PAT in L. japonicus root. LAZY3 transcripts displayed asymmetric distribution at the root tip within hours of gravistimulation, while overexpression of LAZY3 under a constitutive promoter in lazy3 plants rescued the gravitropic response in roots. These data indicate that root gravitropism depends on the presence of LAZY3 but not on its asymmetric expression in root tips. Expression of other LAZY genes in a lazy3 background did not rescue the growth direction of roots, suggesting that the LAZY3 gene plays a distinct role in root gravitropism in L. japonicus.

Root gravity response module guides differential growth determining both root bending and apical hook formation in Arabidopsis.

The apical hook is a transiently formed structure that plays a protective role when the germinating seedling penetrates through the soil towards the surface. Crucial for proper bending is the local auxin maxima, which defines the concave (inner) side of the hook curvature. As no sign of asymmetric auxin distribution has been reported in embryonic hypocotyls prior to hook formation, the question of how auxin asymmetry is established in the early phases of seedling germination remains largely unanswered. Here, we analyzed the auxin distribution and expression of PIN auxin efflux carriers from early phases of germination, and show that bending of the root in response to gravity is the crucial initial cue that governs the hypocotyl bending required for apical hook formation. Importantly, polar auxin transport machinery is established gradually after germination starts as a result of tight root-hypocotyl interaction and a proper balance between abscisic acid and gibberellins.This article has an associated 'The people behind the papers' interview.

Auxin-mediated statolith production for root gravitropism.

Root gravitropism is one of the most important processes allowing plant adaptation to the land environment. Auxin plays a central role in mediating root gravitropism, but how auxin contributes to gravitational perception and the subsequent response are still unclear. Here, we showed that the local auxin maximum/gradient within the root apex, which is generated by the PIN directional auxin transporters, regulates the expression of three key starch granule synthesis genes, SS4, PGM and ADG1, which in turn influence the accumulation of starch granules that serve as a statolith perceiving gravity. Moreover, using the cvxIAA-ccvTIR1 system, we also showed that TIR1-mediated auxin signaling is required for starch granule formation and gravitropic response within root tips. In addition, axr3 mutants showed reduced auxin-mediated starch granule accumulation and disruption of gravitropism within the root apex. Our results indicate that auxin-mediated statolith production relies on the TIR1/AFB-AXR3-mediated auxin signaling pathway. In summary, we propose a dual role for auxin in gravitropism: the regulation of both gravity perception and response.

A role for the auxin precursor anthranilic acid in root gravitropism via regulation of PIN-FORMED protein polarity and relocalisation in Arabidopsis.

distribution of auxin within plant tissues is of great importance for developmental plasticity, including root gravitropic growth. Auxin flow is directed by the subcellular polar distribution and dynamic relocalisation of auxin transporters such as the PIN-FORMED (PIN) efflux carriers, which can be influenced by the main natural plant auxin indole-3-acetic acid (IAA). Anthranilic acid (AA) is an important early precursor of IAA and previously published studies with AA analogues have suggested that AA may also regulate PIN localisation. Using Arabidopsis thaliana as a model species, we studied an AA-deficient mutant displaying agravitropic root growth, treated seedlings with AA and AA analogues and transformed lines to over-produce AA while inhibiting its conversion to downstream IAA precursors. We showed that AA rescues root gravitropic growth in the AA-deficient mutant at concentrations that do not rescue IAA levels. Overproduction of AA affects root gravitropism without affecting IAA levels. Treatments with, or deficiency in, AA result in defects in PIN polarity and gravistimulus-induced PIN relocalisation in root cells. Our results revealed a previously unknown role for AA in the regulation of PIN subcellular localisation and dynamics involved in root gravitropism, which is independent of its better known role in IAA biosynthesis.

AUX1 acts upstream of PIN2 in regulating root gravitropism.

AUX1 and PIN2 auxin transporter are required for the asymmetric distribution of auxin for root gravitropic response. However, the relationship between AUX1 and PIN2 in root gravitropism is unclear. Here, we report that aux1-T mutant show stronger defects in root gravitropism than pin2-T, and aux1-T pin2-T double mutants display similar agravitropic phenotype to aux1-T. The gravity-induced asymmetric distribution of auxin responses could not be established in pin2-T, aux1-T and aux1-T pin2-T mutants; whereas aux1-T pin2-T double mutants showed similar auxin responses to aux1-T mutant. These findings support AUX1 plays a role in root gravitropism upstream of PIN2.

Root Gravitropism: Quantification, Challenges, and Solutions.

Better understanding of root traits such as root angle and root gravitropism will be crucial for development of crops with improved resource use efficiency. This chapter describes a high-throughput, automated image analysis method to trace Arabidopsis (Arabidopsis thaliana) seedling roots grown on agar plates. The method combines a "particle-filtering algorithm with a graph-based method" to trace the center line of a root and can be adopted for the analysis of several root parameters such as length, curvature, and stimulus from original root traces.

Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana.

Plant cells are embedded within cell walls, which provide structural integrity, but also spatially constrain cells, and must therefore be modified to allow cellular expansion. The long-standing acid growth theory postulates that auxin triggers apoplast acidification, thereby activating cell wall-loosening enzymes that enable cell expansion in shoots. Interestingly, this model remains heavily debated in roots, because of both the complex role of auxin in plant development as well as technical limitations in investigating apoplastic pH at cellular resolution. Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a suitable fluorescent pH indicator for assessing apoplastic pH, and thus acid growth, at a cellular resolution in Arabidopsis thaliana roots. Using HPTS, we demonstrate that cell wall acidification triggers cellular expansion, which is correlated with a preceding increase of auxin signaling. Reduction in auxin levels, perception, or signaling abolishes both the extracellular acidification and cellular expansion. These findings jointly suggest that endogenous auxin controls apoplastic acidification and the onset of cellular elongation in roots. In contrast, an endogenous or exogenous increase in auxin levels induces a transient alkalinization of the extracellular matrix, reducing cellular elongation. The receptor-like kinase FERONIA is required for this physiological process, which affects cellular root expansion during the gravitropic response. These findings pinpoint a complex, presumably concentration-dependent role for auxin in apoplastic pH regulation, steering the rate of root cell expansion and gravitropic response.

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.