Hierarchical global and local auxin signals coordinate cellular interdigitation in Arabidopsis.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

PIN2-mediated self-organizing transient auxin flow contributes to auxin maxima at the tip of Arabidopsis cotyledons.

Directional auxin transport and formation of auxin maxima are critical for embryogenesis, organogenesis, pattern formation, and growth coordination in plants, but the mechanisms underpinning the initiation and establishment of these auxin dynamics are not fully understood. Here we show that a self-initiating and -terminating transient auxin flow along the marginal cells (MCs) contributes to the formation of an auxin maximum at the tip of Arabidopsis cotyledon that globally coordinates the interdigitation of puzzle-shaped pavement cells in the cotyledon epidermis. Prior to the interdigitation, indole butyric acid (IBA) is converted to indole acetic acid (IAA) to induce PIN2 accumulation and polarization in the marginal cells, leading to auxin flow toward and accumulation at the cotyledon tip. When IAA levels at the cotyledon tip reaches a maximum, it activates pavement cell interdigitation as well as the accumulation of the IBA transporter TOB1 in MCs, which sequesters IBA to the vacuole and reduces IBA availability and IAA levels. The reduction of IAA levels results in PIN2 down-regulation and cessation of the auxin flow. Hence, our results elucidate a self-activating and self-terminating transient polar auxin transport system in cotyledons, contributing to the formation of localized auxin maxima that spatiotemporally coordinate pavement cell interdigitation.

Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Distinct ADP-ribosylation factor-GTP exchange factors govern the opposite polarity of 2 receptor kinases.

Polarity of plasma membrane proteins is essential for cell morphogenesis and control of cell division and, thus, influences organ and whole plant development. In Arabidopsis (Arabidopsis thaliana) root endodermal cells, 2 transmembrane kinases, INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) and KINASE ON THE INSIDE (KOIN), accumulate at opposite lateral domains. Their polarization is tightly linked to their activities regulating cell division and ground tissue patterning. The polarization of IRK and KOIN relies solely on the secretion of newly synthesized protein. However, the secretion machinery by which their opposite, lateral polarity is achieved remains largely unknown. Here, we show that different sets of ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factors (ARF-GEFs) mediate their secretion. ARF-GEF GNOM-like-1 (GNL1) regulates KOIN secretion to the inner polar domain, thereby directing KOIN sorting early in the secretion pathway. For IRK, combined chemical and genetic analyses showed that the ARG-GEF GNL1, GNOM, and the BREFELDIN A-INHIBITED-GUANINE NUCLEOTIDE-EXCHANGE FACTORs 1 to 4 (BIG1-BIG4) collectively regulate its polar secretion. The ARF-GEF-dependent mechanisms guiding IRK or KOIN lateral polarity were active across different root cell types and functioned regardless of the protein's inner/outer polarity in those cells. Therefore, we propose that specific polar trafficking of IRK and KOIN occurs via distinct mechanisms that are not constrained by cell identity or polar axis and likely rely on individual protein recognition.

Membrane nanodomains: Dynamic nanobuilding blocks of polarized cell growth.

Cell polarity is intimately linked to numerous biological processes, such as oriented plant cell division, particular asymmetric division, cell differentiation, cell and tissue morphogenesis, and transport of hormones and nutrients. Cell polarity is typically initiated by a polarizing cue that regulates the spatiotemporal dynamic of polarity molecules, leading to the establishment and maintenance of polar domains at the plasma membrane. Despite considerable progress in identifying key polarity regulators in plants, the molecular and cellular mechanisms underlying cell polarity formation have yet to be fully elucidated. Recent work suggests a critical role for membrane protein/lipid nanodomains in polarized morphogenesis in plants. One outstanding question is how the spatiotemporal dynamics of signaling nanodomains are controlled to achieve robust cell polarization. In this review, we first summarize the current state of knowledge on potential regulatory mechanisms of nanodomain dynamics, with a special focus on Rho-like GTPases from plants. We then discuss the pavement cell system as an example of how cells may integrate multiple signals and nanodomain-involved feedback mechanisms to achieve robust polarity. A mechanistic understanding of nanodomains' roles in plant cell polarity is still in the early stages and will remain an exciting area for future investigations.

A G-type lectin receptor kinase negatively regulates Arabidopsis immunity against root-knot nematodes.

Root-knot nematodes (Meloidogyne spp., RKN) are responsible for extensive crop losses worldwide. During infection, they penetrate plant roots, migrate between plant cells, and establish feeding sites, known as giant cells, near the root vasculature. Previously, we found that nematode perception and early responses in plants were similar to those of microbial pathogens and required the BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) coreceptor in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). Here, we implemented a reverse genetic screen for resistance or sensitivity to RKN using Arabidopsis T-DNA alleles of genes encoding transmembrane receptor-like kinases to identify additional receptors involved in this process. This screen identified a pair of allelic mutations with enhanced resistance to RKN in a gene we named ENHANCED RESISTANCE TO NEMATODES1 (ERN1). ERN1 encodes a G-type lectin receptor kinase (G-LecRK) with a single-pass transmembrane domain. Further characterization showed that ern1 mutants displayed stronger activation of MAP kinases, elevated levels of the defense marker MYB51, and enhanced H2O2 accumulation in roots upon RKN elicitor treatments. Elevated MYB51 expression and ROS bursts were also observed in leaves of ern1 mutants upon flg22 treatment. Complementation of ern1.1 with 35S- or native promoter-driven ERN1 rescued the RKN infection and enhanced defense phenotypes. Our results indicate that ERN1 is an important negative regulator of immunity.

Distinct mechanisms orchestrate the contra-polarity of IRK and KOIN, two LRR-receptor-kinases controlling root cell division.

In plants, cell polarity plays key roles in coordinating developmental processes. Despite the characterization of several polarly localized plasma membrane proteins, the mechanisms connecting protein dynamics with cellular functions often remain unclear. Here, we introduce a polarized receptor, KOIN, that restricts cell divisions in the Arabidopsis root meristem. In the endodermis, KOIN polarity is opposite to IRK, a receptor that represses endodermal cell divisions. Their contra-polar localization facilitates dissection of polarity mechanisms and the links between polarity and function. We find that IRK and KOIN are recognized, sorted, and secreted through distinct pathways. IRK extracellular domains determine its polarity and partially rescue the mutant phenotype, whereas KOIN's extracellular domains are insufficient for polar sorting and function. Endodermal expression of an IRK/KOIN chimera generates non-cell-autonomous misregulation of root cell divisions that impacts patterning. Altogether, we reveal two contrasting mechanisms determining these receptors' polarity and link their polarity to cell divisions in root tissue patterning.

Confocal Analysis of Arabidopsis Root Cell Divisions in 3D: A Focus on the Endodermis.

The development of multicellular organisms requires coordinated cell divisions for the production of diverse cell types and body plan elaboration and growth. There are two main types of cell divisions: proliferative or symmetric divisions, which produce more cells of a given type, and formative or asymmetric divisions, which produce cells of different types. Because plant cells are surrounded by cell walls, the orientation of plant cell divisions is particularly important in cell fate specification and tissue or organ morphology. The cellular organization of the Arabidopsis thaliana root makes an excellent tool to study how oriented cell division contributes to tissue patterning during organ development. To understand how division plane orientation in a specific genotype or growth condition may impact organ or tissue development, a detailed characterization of cell division orientation is required. Here we describe a confocal microscopy-based, live imaging method for Arabidopsis root tips to examine the 3D orientations of cell division planes and quantify formative, proliferative, and atypical endodermal cell divisions.

Girl power: NORTIA polarization seals pollen tube fate.

How protein dynamics contribute to developmental processes is a critical biological question. In this issue of Developmental Cell, Ju et al. show that subcellular localization of NORTIA in the female gametophyte is required for pollen reception. NORTIA redistribution boosts cues that drive pollen tube bursting, thus promoting male gamete release and fertilization.

A Course-Based Undergraduate Research Experience in CRISPR-Cas9 Experimental Design to Support Reverse Genetic Studies in Arabidopsis thaliana.

Gene-editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene-editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice-weekly meetings over a 5-week period. In the remote-learning version, students performed homology searches, designed guide RNAs (gRNAs) and primers, and learned the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as during closures that have occurred in response to the COVID-19 pandemic. In person, students designed gRNAs, cloned CRISPR-Cas9 constructs, and performed genetic transformation of Arabidopsis thaliana. Students learned how to design effective gRNA pairs targeting their assigned gene with an 86% success rate. Final exams tested students' ability to apply knowledge of an unfamiliar genome database to characterize gene structure and to properly design gRNAs. Average final exam scores of ∼73% and ∼84% for in-person and remote-learning CUREs, respectively, indicated that students met learning outcomes. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of sections. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be adapted readily to other organisms or projects that employ gene editing.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

Auxin-induced signaling protein nanoclustering contributes to cell polarity formation.

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.

A series of dual-reporter vectors for ratiometric analysis of protein abundance in plants.

Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or "target," relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway-compatible vectors termed pRATIO that combine a variety of promoters, fluorescent, and bioluminescent reporters, and 2A "self-cleaving" peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet-I provides the best signal-to-noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal-to-noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin-induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli.

The Arabidopsis Receptor Kinase IRK Is Polarized and Represses Specific Cell Divisions in Roots.

Development of multicellular organisms requires coordination of cell division and differentiation across tissues. In plants, directional signaling, and implicitly cell polarity, is proposed to participate in this coordination; however, mechanistic links between intercellular signaling, cell polarity, and cellular organization remain unclear. Here, we investigate the localization and function of INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) in root development. We find that IRK-GFP localizes to the outer plasma membrane domain in endodermal cells but localizes to different domains in other cell types. Our results suggest that IRK localization is informed locally by adjacent cell types. irk mutants have excess cell divisions in the ground tissue stem cells and endodermis, indicating IRK functions to maintain tissue organization through inhibition of specific cell divisions. We predict that IRK perceives a directional cue that negatively regulates these cell divisions, thus linking intercellular signaling and cell polarity with the control of oriented cell divisions during development.

Asymmetry and cell polarity in root development.

Within living systems, striking juxtapositions in symmetry and asymmetry can be observed and the superficial appearance of symmetric organization often gives way to cellular asymmetries at higher resolution. It is frequently asymmetry and polarity that fascinate and challenge developmental biologists. In multicellular eukaryotes, cell polarity and asymmetry are essential for diverse cellular, tissue, and organismal level function and physiology and are particularly crucial for developmental processes. In plants, where cells are surrounded by rigid cell walls, asymmetric cell divisions are the foundation of pattern formation and differential cell fate specification. Thus, cellular asymmetry is a key feature of plant biology and in the plant root the consequences of these asymmetries are elegantly displayed. Yet despite the frequency of asymmetric (formative) cell divisions, cell/tissue polarity and the proposed roles for directional signaling in these processes, polarly localized proteins, beyond those involved in auxin or nutrient transport, are exceedingly rare. Indeed, although half of the asymmetric cell divisions in root patterning are oriented parallel to the axis of growth, laterally localized proteins directly involved in patterning are largely missing in action. Here, various asymmetric cell divisions and cellular and structural polarities observed in roots are highlighted and discussed in the context of the proposed roles for positional and/or directional signaling in these processes. The importance of directional signaling and the weight given to polarity in the root-shoot axis is contrasted with how little we currently understand about laterally oriented asymmetry and polarity in the root.

Root system patterning: auxin synthesis at the root periphery.

Plasticity in plant form is achieved through differential elaboration of developmental pre-patterns during postembryonic organ development. A new report links the output of the root clock, an oscillatory transcriptional pre-patterning mechanism, with cell-type-specific production of the plant hormone auxin, and identifies a downstream component required for elaboration of the pre-pattern.

Periodic root branching in Arabidopsis requires synthesis of an uncharacterized carotenoid derivative.

In plants, continuous formation of lateral roots (LRs) facilitates efficient exploration of the soil environment. Roots can maximize developmental capacity in variable environmental conditions through establishment of sites competent to form LRs. This LR prepattern is established by a periodic oscillation in gene expression near the root tip. The spatial distribution of competent (prebranch) sites results from the interplay between this periodic process and primary root growth; yet, much about this oscillatory process and the formation of prebranch sites remains unknown. We find that disruption of carotenoid biosynthesis results in seedlings with very few LRs. Carotenoids are further required for the output of the LR clock because inhibition of carotenoid synthesis also results in fewer sites competent to form LRs. Genetic analyses and a carotenoid cleavage inhibitor indicate that an apocarotenoid, distinct from abscisic acid or strigolactone, is specifically required for LR formation. Expression of a key carotenoid biosynthesis gene occurs in a spatially specific pattern along the root's axis, suggesting spatial regulation of carotenoid synthesis. These results indicate that developmental prepatterning of LRs requires an uncharacterized carotenoid-derived molecule. We propose that this molecule functions non-cell-autonomously in establishment of the LR prepattern.