Protocol for analyzing the movement and uptake of isotopically labeled signaling molecule azelaic acid in Arabidopsis.

Understanding the generation, movement, uptake, and perception of mobile defense signals is key for unraveling the systemic resistance programs in flowering plants against pathogens. Here, we present a protocol for analyzing the movement and uptake of isotopically labeled signaling molecule azelaic acid (AZA) in Arabidopsis thaliana. We describe steps to assess 14C-AZA uptake into leaf discs and its movement from local to systemic tissues. We also detail the assay for uptake and movement of 2H-AZA from roots to the shoot. For complete details on the use and execution of this protocol, please refer to Cecchini et al.1,2.

Antigravitropic PIN polarization maintains non-vertical growth in lateral roots.

Lateral roots are typically maintained at non-vertical angles with respect to gravity. These gravitropic setpoint angles are intriguing because their maintenance requires that roots are able to effect growth response both with and against the gravity vector, a phenomenon previously attributed to gravitropism acting against an antigravitropic offset mechanism. Here we show how the components mediating gravitropism in the vertical primary root-PINs and phosphatases acting upon them-are reconfigured in their regulation such that lateral root growth at a range of angles can be maintained. We show that the ability of Arabidopsis lateral roots to bend both downward and upward requires the generation of auxin asymmetries and is driven by angle-dependent variation in downward gravitropic auxin flux acting against angle-independent upward, antigravitropic flux. Further, we demonstrate a symmetry in auxin distribution in lateral roots at gravitropic setpoint angle that can be traced back to a net, balanced polarization of PIN3 and PIN7 auxin transporters in the columella. These auxin fluxes are shifted by altering PIN protein phosphoregulation in the columella, either by introducing PIN3 phosphovariant versions or via manipulation of levels of the phosphatase subunit PP2A/RCN1. Finally, we show that auxin, in addition to driving lateral root directional growth, acts within the lateral root columella to induce more vertical growth by increasing RCN1 levels, causing a downward shift in PIN3 localization, thereby diminishing the magnitude of the upward, antigravitropic auxin flux.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Auxin in Root Development.

Root system architecture is an important determinant of below-ground resource capture and hence overall plant fitness. The plant hormone auxin plays a central role in almost every facet of root development from the cellular to the whole-root-system level. Here, using Arabidopsis as a model, we review the multiple gene signaling networks regulated by auxin biosynthesis, conjugation, and transport that underpin primary and lateral root development. We describe the role of auxin in establishing the root apical meristem and discuss how the tight spatiotemporal regulation of auxin distribution controls transitions between cell division, cell growth, and differentiation. This includes the localized reestablishment of mitotic activity required to elaborate the root system via the production of lateral roots. We also summarize recent discoveries on the effects of auxin and auxin signaling and transport on the control of lateral root gravitropic setpoint angle (GSA), a critical determinant of the overall shape of the root system. Finally, we discuss how environmental conditions influence root developmental plasticity by modulation of auxin biosynthesis, transport, and the canonical auxin signaling pathway.

The Analysis of Gravitropic Setpoint Angle Control in Plants.

The history of research on gravitropism has been largely confined to the primary root-shoot axis and to understanding how the typically vertical orientation observed there is maintained. Many lateral organs are gravitropic too and are often held at specific non-vertical angles relative to gravity. These so-called gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that root and shoot lateral organs are able to effect tropic growth both with and against the gravity vector. This chapter describes methods and considerations relevant to the investigation of mechanisms underlying GSA control.

Kinases and protein motifs required for AZI1 plastid localization and trafficking during plant defense induction.

The proper subcellular localization of defense factors is an important part of the plant immune system. A key component for systemic resistance, lipid transfer protein (LTP)-like AZI1, is needed for the systemic movement of the priming signal azelaic acid (AZA) and a pool of AZI1 exists at the site of AZA production, the plastid envelope. Moreover, after systemic defense-triggering infections, the proportion of AZI1 localized to plastids increases. However, AZI1 does not possess a classical plastid transit peptide that can explain its localization. Instead, AZI1 uses a bipartite N-terminal signature that allows for its plastid targeting. Furthermore, the kinases MPK3 and MPK6, associated with systemic immunity, promote the accumulation of AZI1 at plastids during priming induction. Our results indicate the existence of a mode of plastid targeting possibly related to defense responses.

Peg Biology: Deciphering the Molecular Regulations Involved During Peanut Peg Development.

Peanut or groundnut is one of the most important legume crops with high protein and oil content. The high nutritional qualities of peanut and its multiple usage have made it an indispensable component of our daily life, in both confectionary and therapeutic food industries. Given the socio-economic significance of peanut, understanding its developmental biology is important in providing a molecular framework to support breeding activities. In peanut, the formation and directional growth of a specialized reproductive organ called a peg, or gynophore, is especially relevant in genetic improvement. Several studies have indicated that peanut yield can be improved by improving reproductive traits including peg development. Therefore, we aim to identify unifying principles for the genetic control, underpinning molecular and physiological basis of peg development for devising appropriate strategy for peg improvement. This review discusses the current understanding of the molecular aspects of peanut peg development citing several studies explaining the key mechanisms. Deciphering and integrating recent transcriptomic, proteomic, and miRNA-regulomic studies provide a new perspective for understanding the regulatory events of peg development that participate in pod formation and thus control yield.

An Improved Bioassay to Study Arabidopsis Induced Systemic Resistance (ISR) Against Bacterial Pathogens and Insect Pests.

The plant immune system is essential for plants to perceive and defend against bacterial, fungal and insect pests and pathogens. Induced systemic resistance (ISR) is a systemic immune response that occurs upon root colonization by beneficial microbes. A well-studied model for ISR is the association of specific beneficial strains of Pseudomonas spp. with the reference plant Arabidopsis thaliana. Here, we describe a robust, increased throughput, bioassay to study ISR against the bacterial pathogen Pseudomonas cannabina pv. alisalensis (formerly called Pseudomonas syringae pv. maculicola) strain ES4326 and the herbivore Trichoplusia ni by inoculating Pseudomonas simiae strain WCS417 (formerly called Pseudomonas fluorescens WCS417) on Arabidopsis plants grown in Jiffy-7® peat pellets. While most commonly used for Pseudomonas-triggered ISR on Arabidopsis, this assay is effective for diverse rhizosphere bacterial strains, plant species, pathogens and herbivores.

Underground Azelaic Acid-Conferred Resistance to Pseudomonas syringae in Arabidopsis.

Local interactions between individual plant organs and diverse microorganisms can lead to whole plant immunity via the mobilization of defense signals. One such signal is the plastid lipid-derived oxylipin azelaic acid (AZA). Arabidopsis lacking AZI1 or EARLI1, related lipid transfer family proteins, exhibit reduced AZA transport among leaves and cannot mount systemic immunity. AZA has been detected in roots as well as leaves. Therefore, the present study addresses the effects on plants of AZA application to roots. AZA but not the structurally related suberic acid inhibits root growth when directly in contact with roots. Treatment of roots with AZA also induces resistance to Pseudomonas syringae in aerial tissues. These effects of AZA on root growth and disease resistance depend, at least partially, on AZI1 and EARLI1. AZI1 in roots localizes to plastids, similar to its known location in leaves. Interestingly, kinases previously shown to modify AZI1 in vitro, MPK3 and MPK6, are also needed for AZA-induced root-growth inhibition and aboveground immunity. Finally, deuterium-labeled AZA applied to the roots does not move to aerial tissues. Thus, AZA application to roots triggers systemic immunity through an AZI1/EARLI1/MPK3/MPK6-dependent pathway and AZA effects may involve one or more additional mobile signals.

The developmental and environmental regulation of gravitropic setpoint angle in Arabidopsis and bean.

Root and shoot branches are major determinants of plant form and critical for the effective capture of resources below and above ground. These branches are often maintained at specific angles with respect to gravity, known as gravitropic set point angles (GSAs). We have previously shown that the mechanism permitting the maintenance of non-vertical GSAs is highly auxin-dependent and here we investigate the developmental and environmental regulation of root and shoot branch GSA. We show that nitrogen and phosphorous deficiency have opposing, auxin signalling-dependent effects on lateral root GSA in Arabidopsis: while low nitrate induces less vertical lateral root GSA, phosphate deficiency results in a more vertical lateral root growth angle, a finding that contrasts with the previously reported growth angle response of bean adventitious roots. We find that this root-class-specific discrepancy in GSA response to low phosphorus is mirrored by similar differences in growth angle response to auxin treatment between these root types. Finally we show that both shaded, low red/far-red light conditions and high temperature induce more vertical growth in Arabidopsis shoot branches. We discuss the significance of these findings in the context of efforts to improve crop performance via the manipulation of root and shoot branch growth angle.

Analysis of gravitropic setpoint angle control in Arabidopsis.

The history of research on gravitropism has been largely confined to the primary root-shoot axis and to understanding how the typically vertical orientation observed there is maintained. Many lateral organs are gravitropic too and are often held at specific non-vertical angles relative to gravity. These so-called gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that root and shoot lateral organs are able to effect tropic growth both with and against the gravity vector. This chapter describes methods and considerations relevant to the investigation of mechanisms underlying GSA control.

Shoot and root branch growth angle control-the wonderfulness of lateralness.

The overall shape of plants, the space they occupy above and below ground, is determined principally by the number, length, and angle of their lateral branches. The function of these shoot and root branches is to hold leaves and other organs to the sun, and below ground, to provide anchorage and facilitate the uptake of water and nutrients. While in some respects lateral roots and shoots can be considered mere iterations of the primary root-shoot axis, in others there are fundamental differences in their biology, perhaps most conspicuously in the regulation their angle of growth. Here we discuss recent advances in the understanding of the control of branch growth angle, one of the most important but least understood components of the wonderful diversity of plant form observed throughout nature.

Auxin controls gravitropic setpoint angle in higher plant lateral branches.

Lateral branches in higher plants are often maintained at specific angles with respect to gravity, a quantity known as the gravitropic setpoint angle (GSA) [1]. Despite the importance of GSA control as a fundamental determinant of plant form, the mechanisms underlying gravity-dependent angled growth are not known. Here we address the central questions of how stable isotropic growth of a branch at a nonvertical angle is maintained and of how the value of that angle is set. We show that nonvertical lateral root and shoot branches are distinguished from the primary axis by the existence of an auxin-dependent antigravitropic offset mechanism that operates in tension with gravitropic response to generate angled isotropic growth. Further, we show that the GSA of lateral roots and shoots is dependent upon the magnitude of the antigravitropic offset component. Finally, we show that auxin specifies GSA values dynamically throughout development by regulating the magnitude of the antigravitropic offset component via TIR1/AFB-Aux/IAA-ARF-dependent auxin signaling within the gravity-sensing cells of the root and shoot. The involvement of auxin in controlling GSA is yet another example of auxin's remarkable capacity to self-organize in development [2] and provides a conceptual framework for understanding the specification of GSA throughout nature.