A dual function of the IDA peptide in regulating cell separation and modulating plant immunity at the molecular level.

The abscission of floral organs and emergence of lateral roots in Arabidopsis is regulated by the peptide ligand inflorescence deficient in abscission (IDA) and the receptor protein kinases HAESA (HAE) and HAESA-like 2 (HSL2). During these cell separation processes, the plant induces defense-associated genes to protect against pathogen invasion. However, the molecular coordination between abscission and immunity has not been thoroughly explored. Here, we show that IDA induces a release of cytosolic calcium ions (Ca2+) and apoplastic production of reactive oxygen species, which are signatures of early defense responses. In addition, we find that IDA promotes late defense responses by the transcriptional upregulation of genes known to be involved in immunity. When comparing the IDA induced early immune responses to known immune responses, such as those elicited by flagellin22 treatment, we observe both similarities and differences. We propose a molecular mechanism by which IDA promotes signatures of an immune response in cells destined for separation to guard them from pathogen attack.

A multifaceted kinase axis regulates plant organ abscission through conserved signaling mechanisms.

Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.

Dissection of the IDA promoter identifies WRKY transcription factors as abscission regulators in Arabidopsis.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

Adaptive protein evolution through length variation of short tandem repeats in Arabidopsis.

Intrinsically disordered protein regions are of high importance for biotic and abiotic stress responses in plants. Tracts of identical amino acids accumulate in these regions and can vary in length over generations because of expansions and retractions of short tandem repeats at the genomic level. However, little attention has been paid to what extent length variation is shaped by natural selection. By environmental association analysis on 2514 length variable tracts in 770 whole-genome sequenced Arabidopsis thaliana, we show that length variation in glutamine and asparagine amino acid homopolymers, as well as in interaction hotspots, correlate with local bioclimatic habitat. We determined experimentally that the promoter activity of a light-stress gene depended on polyglutamine length variants in a disordered transcription factor. Our results show that length variations affect protein function and are likely adaptive. Length variants modulating protein function at a global genomic scale has implications for understanding protein evolution and eco-evolutionary biology.

The Cell Fate Controlling CLE40 Peptide Requires CNGCs to Trigger Highly Localized Ca2+ Transients in Arabidopsis thaliana Root Meristems.

Communication between plant cells and their biotic environment largely depends on the function of plasma membrane localized receptor-like kinases (RLKs). Major players in this communication within root meristems are secreted peptides, including CLAVATA3/EMBRYO SURROUNDING REGION40 (CLE40). In the distal root meristem, CLE40 acts through the RLK ARABIDOPSIS CRINKLY4 (ACR4) and the leucine-rich repeat (LRR) RLK CLAVATA1 (CLV1) to promote cell differentiation. In the proximal meristem, CLE40 signaling requires the LRR receptor-like protein CLAVATA2 (CLV2) and the membrane localized pseudokinase CORYNE (CRN) and serves to inhibit cell differentiation. The molecular components that act immediately downstream of the CLE40-activated receptors are not yet known. Here, we show that active CLE40 signaling triggers the release of intracellular Ca2+ leading to increased cytosolic Ca2+ concentration ([Ca2+]cyt) in a small subset of proximal root meristem cells. This rise in [Ca2+]cyt depends on the CYCLIC NUCLEOTIDE GATED CHANNELS (CNGCs) 6 and 9 and on CLV1. The precise function of changes in [Ca2+]cyt is not yet known but might form a central part of a fine-tuned response to CLE40 peptide that serves to integrate root meristem growth with stem cell fate decisions and initiation of lateral root primordia.

Length variation in short tandem repeats affects gene expression in natural populations of Arabidopsis thaliana.

The genetic basis for the fine-tuned regulation of gene expression is complex and ultimately influences the phenotype and thus the local adaptation of natural populations. Short tandem repeats (STRs) consisting of repetitive DNA motifs have been shown to regulate gene expression. STRs are variable in length within a population and serve as a heritable, but semi-reversible, reservoir of standing genetic variation. For sessile organisms, such as plants, STRs could be of major importance in fine-tuning gene expression as a response to a shifting local environment. Here, we used a transcriptome dataset from natural accessions of Arabidopsis thaliana to investigate population-wide gene expression patterns in light of genome-wide STR variation. We empirically modeled gene expression as a response to the STR length within and around the gene and demonstrated that an association between gene expression and STR length variation is unequivocally present in the sampled population. To support our model, we explored the promoter activity in a transcriptional regulator involved in root hair formation and provided experimentally determined causality between coding sequence length variation and promoter activity. Our results support a general link between gene expression variation and STR length variation in A. thaliana.

Emerging mechanisms to fine-tune receptor kinase signaling specificity.

Organisms need to constantly inform their cellular machinery about the biochemical and physical status of their surroundings to adapt and thrive. While some external signals are also sensed intracellularly, a considerable share of external information is registered already at the plasma membrane (PM). Receptor kinases (RKs) are crucial for plant cells to integrate such cues from the environment, from microbes, or from other cells to coordinate their physiological response and their development. Early studies on RK signaling depicted the path from external signal to internal response in a linear fashion, but recent findings show that these cellular information highways are highly interconnected and pass signals through molecular intersections. In this review, we first discuss how individual RKs simultaneously contribute to the transduction and deconvolution of a multitude of signals by controlled assembly into diverse RK complexes, exemplified by FERONIA signaling versatility. We then elaborate on how cells can exert highly localized control over the assembly, interaction and composition of such complexes in order to attain essential cellular output specificity.

Look Closely, the Beautiful May Be Small: Precursor-Derived Peptides in Plants.

During the past decade, a flurry of research focusing on the role of peptides as short- and long-distance signaling molecules in plant cell communication has been undertaken. Here, we focus on peptides derived from nonfunctional precursors, and we address several key questions regarding peptide signaling. We provide an overview of the regulatory steps involved in producing a biologically active peptide ligand that can bind its corresponding receptor(s) and discuss how this binding and subsequent activation lead to specific cellular outputs. We discuss different experimental approaches that can be used to match peptide ligands with their receptors. Lastly, we explore how peptides evolved from basic signaling units regulating essential processes in plants to more complex signaling systems as new adaptive traits developed and how nonplant organisms exploit this signaling machinery by producing peptide mimics.

The dynamics of root cap sloughing in Arabidopsis is regulated by peptide signalling.

The root cap protects the stem cell niche of angiosperm roots from damage. In Arabidopsis, lateral root cap (LRC) cells covering the meristematic zone are regularly lost through programmed cell death, while the outermost layer of the root cap covering the tip is repeatedly sloughed. Efficient coordination with stem cells producing new layers is needed to maintain a constant size of the cap. We present a signalling pair, the peptide IDA-LIKE1 (IDL1) and its receptor HAESA-LIKE2 (HSL2), mediating such communication. Live imaging over several days characterized this process from initial fractures in LRC cell files to full separation of a layer. Enhanced expression of IDL1 in the separating root cap layers resulted in increased frequency of sloughing, balanced with generation of new layers in a HSL2-dependent manner. Transcriptome analyses linked IDL1-HSL2 signalling to the transcription factors BEARSKIN1/2 and genes associated with programmed cell death. Mutations in either IDL1 or HSL2 slowed down cell division, maturation and separation. Thus, IDL1-HSL2 signalling potentiates dynamic regulation of the homeostatic balance between stem cell division and sloughing activity.

Abscission in plants.

Olsson and Butenko introduce the topic of abscission in plants.

Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors.

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

Quantitative Analysis of Floral Organ Abscission in Arabidopsis Via a Petal Breakstrength Assay.

Petal breakstrength (pBS) is a method to study floral organ abscission by quantitating the force required to pull a petal from the receptacle. However, it is only well established in some labs and used in a subset of abscission studies. Here, we describe the mechanism and operation of the pBS meter, as well as detailed measurement and further data analysis. We show that it is a powerful tool to detect early or delayed floral organ abscission in mutant or transgenic plants, which is not easily detected by phenotypic investigation.

Visualizing Morphological Changes of Abscission Zone Cells in Arabidopsis by Scanning Electron Microscope.

Scanning electron microscope (SEM) is a type of electron microscope which produces detailed images of surface structures. It has been widely used in plants and animals to study cellular structures. Here, we describe a detailed protocol to prepare samples of floral abscission zones (AZs) for SEM, as well as further image analysis. We show that it is a powerful tool to detect morphologic changes at the cellular level during the course of abscission in wild-type plants and to establish the details of phenotypic alteration in abscission mutants.

Chemiluminescence-Based Detection of Peptide Activity and Peptide-Receptor Binding in Plants.

In living organisms, physical interaction of ligand molecules with their cognate receptors is an indispensable requirement for the initiation of cellular signaling pathways. To technically prove the biochemical interaction of ligands with their corresponding receptor, a biologically active but labeled peptide is required. Easily scorable bioassays, such as the production of reactive oxygen species, can be used to quantify the activity of a peptide. By using chemiluminescent probes, such as acridinium esters, as conjugates to label peptide ligands of interest, quantitative measurements of ligand-receptor binding can be performed in standard luminometers. Here we describe how this binding approach can be used to reveal peptide ligand-receptor binding in plant material.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

Mechanistic insight into a peptide hormone signaling complex mediating floral organ abscission.

Plants constantly renew during their life cycle and thus require to shed senescent and damaged organs. Floral abscission is controlled by the leucine-rich repeat receptor kinase (LRR-RK) HAESA and the peptide hormone IDA. It is unknown how expression of IDA in the abscission zone leads to HAESA activation. Here we show that IDA is sensed directly by the HAESA ectodomain. Crystal structures of HAESA in complex with IDA reveal a hormone binding pocket that accommodates an active dodecamer peptide. A central hydroxyproline residue anchors IDA to the receptor. The HAESA co-receptor SERK1, a positive regulator of the floral abscission pathway, allows for high-affinity sensing of the peptide hormone by binding to an Arg-His-Asn motif in IDA. This sequence pattern is conserved among diverse plant peptides, suggesting that plant peptide hormone receptors may share a common ligand binding mode and activation mechanism.

The IDA Peptide Controls Abscission in Arabidopsis and Citrus.

Organ abscission is an important process in plant development and reproduction. During abscission, changes in cellular adhesion of specialized abscission zone cells ensure the detachment of infected organs or those no longer serving a function to the plant. In addition, abscission also plays an important role in the release of ripe fruits. Different plant species display distinct patterns and timing of organ shedding, most likely adapted during evolution to their diverse life styles. However, it appears that key regulators of cell separation may have conserved function in different plant species. Here, we investigate the functional conservation of the citrus ortholog of the Arabidopsis peptide ligand INFLORESCENCE DEFICIENT IN ABSCISSION (AtIDA), controlling floral organ abscission. We discuss the possible implications of modifying the citrus IDA ortholog for citrus fruit production.

Antagonistic peptide technology for functional dissection of CLE peptides revisited.

In the Arabidopsis thaliana genome, over 1000 putative genes encoding small, presumably secreted, signalling peptides can be recognized. However, a major obstacle in identifying the function of genes encoding small signalling peptides is the limited number of available loss-of-function mutants. To overcome this, a promising new tool, antagonistic peptide technology, was recently developed. Here, this antagonistic peptide technology was tested on selected CLE peptides and the related IDA peptide and its usefulness in the context of studies of peptide function discussed. Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines. However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful. This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

Modulation of Arabidopsis and monocot root architecture by CLAVATA3/EMBRYO SURROUNDING REGION 26 peptide.

Plant roots are important for a wide range of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface with the soil environment. Several small signalling peptides and receptor kinases have been shown to affect primary root growth, but very little is known about their role in lateral root development. In this context, the CLE family, a group of small signalling peptides that has been shown to affect a wide range of developmental processes, were the focus of this study. Here, the expression pattern during lateral root initiation for several CLE family members is explored and to what extent CLE1, CLE4, CLE7, CLE26, and CLE27, which show specific expression patterns in the root, are involved in regulating root architecture in Arabidopsis thaliana is assessed. Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling. In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed. Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented.

Beyond the meristems: similarities in the CLAVATA3 and INFLORESCENCE DEFICIENT IN ABSCISSION peptide mediated signalling pathways.

Plants form new organs throughout their lives; this requires a balance between cell proliferation and differentiation, and between the generation and loss of organs. To do this, plants must maintain a population of stem cells within the meristems, and at the same time, closely control the identity and position of cells at the meristem boundaries as they differentiate to new leaf or flower primordia. Once developed, organs may need to be shed, either as a controlled developmental decision-such as floral abscission after pollination, or as a response to disease, environmental stress, and predators. Cell wall degradation at specialized abscission zone (AZ) cells needs to occur for this to take place, but since there is little cell rearrangement in plants, cell separation events are also important for plant architecture. In this Opinion paper we discuss the role of two peptide ligand signalling systems that control stem cell homeostasis and cell separation, respectively. We draw parallels between the signalling pathways and explore on the commonalities of the downstream components activated and controlled by the signalling peptides. We provide evidence for AZ cells having a meristem identity and discuss the role of identical KNOTTED-LIKE HOMEOBOX (KNOX) transcription factors in meristem maintenance and abscission. Lastly we explore the evolutionary relationship between the pathways.