An updated model of shoot apical meristem regulation by ERECTA family and CLAVATA3 signaling pathways in Arabidopsis.

The shoot apical meristem (SAM) gives rise to the aboveground organs of plants. The size of the SAM is relatively constant due to the balance between stem cell replenishment and cell recruitment into new organs. In angiosperms, the transcription factor WUSCHEL (WUS) promotes stem cell proliferation in the central zone of the SAM. WUS forms a negative feedback loop with a signaling pathway activated by CLAVATA3 (CLV3). In the periphery of the SAM, the ERECTA family receptors (ERfs) constrain WUS and CLV3 expression. Here, we show that four ligands of ERfs redundantly inhibit the expression of these two genes. Transcriptome analysis confirmed that WUS and CLV3 are the main targets of ERf signaling and uncovered new ones. Analysis of promoter reporters indicated that the WUS expression domain mostly overlaps with the CLV3 domain and does not shift along the apical-basal axis in clv3 mutants. Our three-dimensional mathematical model captured gene expression distributions at the single-cell level under various perturbed conditions. Based on our findings, CLV3 regulates cellular levels of WUS mostly through autocrine signaling, and ERfs regulate the spatial expression of WUS, preventing its encroachment into the peripheral zone.

Initiation of aboveground organ primordia depends on combined action of auxin, ERECTA family genes, and PINOID.

Leaves and flowers are produced by the shoot apical meristem (SAM) at a certain distance from its center, a process that requires the hormone auxin. The amount of auxin and the pattern of its distribution in the initiation zone determine the size and spatial arrangement of organ primordia. Auxin gradients in the SAM are formed by PIN-FORMED (PIN) auxin efflux carriers whose polar localization in the plasma membrane depends on the protein kinase PINOID (PID). Previous work determined that ERECTA (ER) family genes (ERfs) control initiation of leaves. ERfs are plasma membrane receptors that enable cell-to-cell communication by sensing extracellular small proteins from the EPIDERMAL PATTERNING FACTOR/EPF-LIKE (EPF/EPFL) family. Here, we investigated whether ERfs regulate initiation of organs by altering auxin distribution or signaling in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological data suggested that ERfs do not regulate organogenesis through PINs while transcriptomics data showed that ERfs do not alter primary transcriptional responses to auxin. Our results indicated that in the absence of ERf signaling the peripheral zone cells inefficiently initiate leaves in response to auxin signals and that increased accumulation of auxin in the er erecta-like1 (erl1) erl2 SAM can partially rescue organ initiation defects. We propose that both auxin and ERfs are essential for leaf initiation and that they have common downstream targets. Genetic data also indicated that the role of PID in initiation of cotyledons and leaves cannot be attributed solely to regulation of PIN polarity and PID is likely to have other functions in addition to regulation of auxin distribution.

ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem.

The shoot apical meristem (SAM) is a reservoir of stem cells that gives rise to all post-embryonic above-ground plant organs. The size of the SAM remains stable over time owing to a precise balance of stem cell replenishment versus cell incorporation into organ primordia. The WUSCHEL (WUS)/CLAVATA (CLV) negative feedback loop is central to SAM size regulation. Its correct function depends on accurate spatial expression of WUS and CLV3 A signaling pathway, consisting of ERECTA family (ERf) receptors and EPIDERMAL PATTERNING FACTOR LIKE (EPFL) ligands, restricts SAM width and promotes leaf initiation. Although ERf receptors are expressed throughout the SAM, EPFL ligands are expressed in its periphery. Our genetic analysis of Arabidopsis demonstrated that ERfs and CLV3 synergistically regulate the size of the SAM, and wus is epistatic to ERf genes. Furthermore, activation of ERf signaling with exogenous EPFLs resulted in a rapid decrease of CLV3 and WUS expression. ERf-EPFL signaling inhibits expression of WUS and CLV3 in the periphery of the SAM, confining them to the center. These findings establish the molecular mechanism for stem cell positioning along the radial axis.

A mathematical model for understanding synergistic regulations and paradoxical feedbacks in the shoot apical meristem.

The shoot apical meristem (SAM) is the primary stem cell niche in plant shoots. Stem cells in the SAM are controlled by an intricate regulatory network, including negative feedback between WUSCHEL (WUS) and CLAVATA3 (CLV3). Recently, we identified a group of signals, Epidermal Patterning Factor-Like (EPFL) proteins, that are produced at the peripheral region and are important for SAM homeostasis. Here, we present a mathematical model for the SAM regulatory network. The model revealed that the SAM uses EPFL and signals such as HAIRY MERISTEM from the middle in a synergistic manner to constrain both WUS and CLV3. We found that interconnected negative and positive feedbacks between WUS and CLV3 ensure stable WUS expression in the SAM when facing perturbations, and the positive feedback loop also maintains distinct cell populations containing WUS on and CLV3 on cells in the apical-basal direction. Furthermore, systematic perturbations of the parameters revealed a tradeoff between optimizations of multiple patterning features. Our results provide a holistic view of the regulation of SAM patterning in multiple dimensions. They give insights into how Arabidopsis integrates signals from lateral and apical-basal axes to control the SAM patterning, and they shed light into design principles that may be widely useful for understanding regulatory networks of stem cell niche.

EPFL Signals in the Boundary Region of the SAM Restrict Its Size and Promote Leaf Initiation.

The shoot apical meristem (SAM) enables the formation of new organs throughout the life of a plant. ERECTA family (ERf) receptors restrict SAM size and promote initiation of leaves while simultaneously supporting establishment of correct phyllotaxy. In the epidermis and during organ elongation ERf activity is regulated by a family of Epidermal Patterning Factor-Like (EPFL) secreted Cys-rich small proteins. Here we show that ERfs play a critical role in communication between the SAM leaf boundary and the central zone in Arabidopsis (Arabidopsis thaliana). Ectopic expression of ERECTA in the central zone using the CLAVATA3 promoter is sufficient to restrict meristem size and promote leaf initiation. Genetic analysis demonstrated that four putative ligands: EPFL1, EPFL2, EPFL4, and EPFL6 function redundantly in the SAM. These genes are expressed at the SAM-leaf boundary and in the peripheral zone. Previously EPFL4 and EPFL6 have been linked with elongation of aboveground organs. Here we demonstrate that EPFL1 and EPFL2 promote organ elongation as well. In addition, we show that expression of ERECTA in the central zone of the SAM has a strong impact on elongation of internodes and pedicels and growth of leaves. These results suggest that ERfs can stimulate organ growth cell nonautonomously.

A receptor-like protein acts as a specificity switch for the regulation of stomatal development.

Stomata are microscopic openings that allow for the exchange of gases between plants and the environment. In Arabidopsis, stomatal patterning is specified by the ERECTA family (ERf) receptor kinases (RKs), the receptor-like protein (RLP) TOO MANY MOUTHS (TMM), and EPIDERMAL PATTERNING FACTOR (EPF) peptides. Here we show that TMM and ER or ER-LIKE1 (ERL1) form constitutive complexes, which recognize EPF1 and EPF2, but the single ERfs do not. TMM interaction with ERL1 creates a binding pocket for recognition of EPF1 and EPF2, indicating that the constitutive TMM-ERf complexes function as the receptors of EPF1 and EPF2. EPFL9 competes with EPF1 and EPF2 for binding to the ERf-TMM complex. EPFL4 and EPFL6, however, are recognized by the single ERfs without the requirement of TMM. In contrast to EPF1,2, the interaction of EPFL4,6 with an ERf is greatly reduced in the presence of TMM. Taken together, our data demonstrate that TMM dictates the specificity of ERfs for the perception of different EPFs, thus functioning as a specificity switch for the regulation of the activities of ERfs.

Identification of critical functional residues of receptor-like kinase ERECTA.

In plants, extracellular signals are primarily sensed by plasma membrane-localized receptor-like kinases (RLKs). ERECTA is a leucine-rich repeat RLK that together with its paralogs ERECTA-like 1 (ERL1) and ERL2 regulates multiple aspects of plant development. ERECTA forms complexes with a range of co-receptors and senses secreted cysteine-rich small proteins from the EPF/EPFL family. Currently the mechanism of the cytoplasmic domain activation and transmission of the signal by ERECTA is unclear. To gain a better understanding we performed a structure-function analysis by introducing altered ERECTA genes into erecta and erecta erl1 erl2 mutants. These experiments indicated that ERECTA's ability to phosphorylate is functionally significant, and that while the cytoplasmic juxtamembrane domain is important for ERECTA function, the C-terminal tail is not. An analysis of multiple putative phosphorylation sites identified four amino acids in the activation segment of the kinase domain as functionally important. Homology of those residues to functionally significant amino acids in multiple other plant RLKs emphasizes similarities in RLK function. Specifically, our data predicts Thr812 as a primary site of phosphor-activation and potential inhibitory phosphorylation of Tyr815 and Tyr820. In addition, our experiments suggest that there are differences in the molecular mechanism of ERECTA function during regulation of stomata development and in elongation of above-ground organs.

A Mutation in the Catalytic Subunit of the Glycosylphosphatidylinositol Transamidase Disrupts Growth, Fertility, and Stomata Formation.

GPI-anchored proteins (GPI-APs) are essential for plant growth and development; knockout mutations in enzymes responsible for anchor biosynthesis or attachment are gametophyte or embryo lethal. In a genetic screen targeted to identify genes regulating stomata formation, we discovered a missense mutation in the Arabidopsis (Arabidopsis thaliana) homolog of GPI8/PIG-K, a Cys protease that transfers an assembled GPI anchor to proteins. The Arabidopsis genome has a single copy of AtGPI8, and the atgpi8-1 mutation reduces the efficiency of this enzyme, leading to reduced accumulation of GPI-anchored proteins. While the atgpi8-1 mutation strongly disrupts plant growth, it is not lethal. Phenotypic analysis of atgpi8-1 mutants suggests that GPI-APs are important for root and shoot growth, stomata formation, apical dominance, transition to flowering, and male gametophyte viability. In addition, atgpi8-1 mutants accumulate higher levels of callose and have reduced plasmodesmata permeability. Genetic interactions of atgpi8-1 with mutations in ERECTA family (ERf) genes suggest the existence of a GPI-AP in a branch of the ERf signaling pathway that regulates stomata formation. Activation of the ERf signal transduction cascade by constitutively active YODA rescues stomata clustering in atgpi8-1, indicating that a GPI-AP functions upstream of the MAP kinase cascade. TOO MANY MOUTHS (TMM) is a receptor-like protein that is able to form heterodimers with ERfs. Our analysis demonstrates that tmm-1 is epistatic to atgpi8-1, indicating that either TMM is a GPI-AP or there is another GPI-AP regulating stomata development whose function is dependent upon TMM.

Carbon Nanofiber Arrays: A Novel Tool for Microdelivery of Biomolecules to Plants.

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, a common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5-500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants.

The Role of LORELEI in Pollen Tube Reception at the Interface of the Synergid Cell and Pollen Tube Requires the Modified Eight-Cysteine Motif and the Receptor-Like Kinase FERONIA.

In angiosperms, pollen tube reception by the female gametophyte is required for sperm release and double fertilization. In Arabidopsis thaliana lorelei (lre) mutants, pollen tube reception fails in most female gametophytes, which thus remain unfertilized. LRE encodes a putative glycosylphosphatidylinositol (GPI)-anchored surface protein with a modified eight-cysteine motif (M8CM). LRE fused to citrine yellow fluorescent protein (LRE-cYFP) remains functional and localizes to the synergid plasma membrane-rich filiform apparatus, the first point of contact between the pollen tube and the female gametophyte. Structure-function analysis using LRE-cYFP showed that the role of LRE in pollen tube reception requires the M8CM, but not the domains required for GPI anchor addition. Consistently, LRE-cYFP-TM, where GPI anchor addition domains were replaced with a single-pass transmembrane domain, fully complemented the pollen tube reception defect in lre-7 female gametophytes. Ectopically expressed and delivered LRE-cYFP from pollen tubes could non-cell-autonomously complement the pollen tube reception defect in lre female gametophytes, only if they expressed FERONIA. Additionally, pollen tube-expressing LRE variants lacking domains critical for GPI anchor addition also rescued lre female gametophyte function. Therefore, LRE and FERONIA jointly function in pollen tube reception at the interface of the synergid cell and pollen tube.

ERECTA family genes regulate development of cotyledons during embryogenesis.

Receptor-like kinases are important regulators of plant growth. Often a single receptor is involved in regulation of multiple developmental processes in a variety of tissues. ERECTA family (ERf) receptors have previously been linked with stomata development, above-ground organ elongation, shoot apical meristem function, flower differentiation and biotic/abiotic stresses. Here we explore the role of these genes during embryogenesis. ERfs are expressed in the developing embryo, where their expression is progressively limited to the upper half of the embryo. During embryogenesis ERfs redundantly stimulate the growth of cotyledons by promoting cell proliferation and inhibiting premature stomata differentiation.

Regulation of floral patterning and organ identity by Arabidopsis ERECTA-family receptor kinase genes.

Due to the lack of cell migration, plant organogenesis relies on coordinated cell proliferation, cell growth, and differentiation. A flower possesses a complex structure, with sepals and petals constituting the perianth, and stamens and pistils where male and female gametophytes differentiate. While advances have been made in our understanding of gene regulatory networks controlling flower development, relatively little is known of how cell-cell coordination influences floral organ specification. The Arabidopsis ERECTA (ER)-family receptor kinases, ER, ER-LIKE1 (ERL1), and ERL2, regulate inflorescence architecture, organ shape, and epidermal stomatal patterning. Here it is reported that ER-family genes together regulate floral meristem organization and floral organ identity. The stem cell marker CLAVATA3 exhibits misplaced expression in the floral meristems of the er erl1 erl2 mutant. Strikingly, homeotic conversion of sepals to carpels was observed in er erl1 erl2 flowers. Consistently, ectopic expression of AGAMOUS, which determines carpel identity, was detected in er erl1 erl2 flower primordia. Among the known downstream components of ER-family receptor kinases in stomatal patterning, YODA (YDA) is also required for proper floral patterning. YDA and the ER-family show complex, synergistic genetic interactions: er erl1 erl2 yda quadruple mutant plants become extremely small, callus-like masses. While a constitutively active YDA fully rescues stomatal clustering in er erl1 erl2, it only partially rescues er erl1 erl2 flower defects. The study suggests that ER-family signalling is crucial for ensuring proper expression domains of floral meristem and floral organ identity determinants, and further implies the existence of a non-canonical downstream pathway.

Diverse roles of ERECTA family genes in plant development.

Multiple receptor-like kinases (RLKs) enable intercellular communication that coordinates growth and development of plant tissues. ERECTA family receptors (ERfs) are an ancient family of leucine-rich repeat RLKs that in Arabidopsis consists of three genes: ERECTA, ERL1, and ERL2. ERfs sense secreted cysteine-rich peptides from the EPF/EPFL family and transmit the signal through a MAP kinase cascade. This review discusses the functions of ERfs in stomata development, in regulation of longitudinal growth of aboveground organs, during reproductive development, and in the shoot apical meristem. In addition the role of ERECTA in plant responses to biotic and abiotic factors is examined. Elena D. Shpak (Corresponding author).

ERECTA family genes regulate auxin transport in the shoot apical meristem and forming leaf primordia.

Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.

Modification of tomato growth by expression of truncated ERECTA protein from Arabidopsis thaliana.

ERECTA family genes encode leucine-rich repeat receptor-like kinases that control multiple aspects of plant development such as elongation of aboveground organs, leaf initiation, development of flowers, and epidermis differentiation. These receptors have also been implicated in responses to biotic and abiotic stress, probably as a consequence of their involvement in regulation of plant architecture. Here, ERECTA signalling in tomatoes (Solanum lycopersicum) was manipulated by expressing truncated ERECTA protein (AtΔKinase) from Arabidopsis using two different promoters. In Arabidopsis, this protein functions in a dominant-negative manner, disrupting signalling of the whole ERECTA gene family. Expression of AtΔKinase under a constitutive 35S promoter dramatically reduced vegetative growth and led to the formation of fruits with a reduced seed set. Similarly, expression of AtΔKinase under its own promoter resulted in transgenic tomato plants with diminished growth, a reduced number of leaves, changed flowering time, and slightly increased stomata density. The transgenic plants also exhibited increased tolerance to water deficit stress, at least partially due to their diminished surface area. These phenotypes of the transgenic plants were the result of ERECTA signalling disruption at the protein level, as the expression of two endogenous tomato ERECTA family genes was not suppressed. These results demonstrate the significance of ERECTA family genes for development and stress responses in tomato and suggest that truncated ERECTA can be used to manipulate the growth of crop species.

Patterns of cell division, cell differentiation and cell elongation in epidermis and cortex of Arabidopsis pedicels in the wild type and in erecta.

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.

Regulation of plasmodesmatal permeability and stomatal patterning by the glycosyltransferase-like protein KOBITO1.

The differentiation of stomata provides a convenient model for studying pattern formation in plant tissues. Stomata formation is induced by a set of basic helix-loop-helix transcription factors and inhibited by a signal transduction pathway initiated by TOO MANY MOUTHS (TMM) and ERECTA family (ERf) receptors. The formation of a proper stomata pattern is also dependent upon the restriction of symplastic movement of basic helix-loop-helix transcription factors into neighboring cells, especially in the backgrounds where the function of the TMM/ERf signaling pathway is compromised. Here, we describe a novel mutant of KOBITO1 in Arabidopsis (Arabidopsis thaliana). The kob1-3 mutation leads to the formation of stomata clusters in the erl1 erl2 background but not in the wild type. Cell-to-cell mobility assays demonstrated an increase in intercellular protein trafficking in kob1-3, including increased diffusion of SPEECHLESS, suggesting that the formation of stomata clusters is due to an escape of cell fate-specifying factors from stomatal lineage cells. While plasmodesmatal permeability is increased in kob1-3, we did not detect drastic changes in callose accumulation at the neck regions of the plasmodesmata. Previously, KOBITO1 has been proposed to function in cellulose biosynthesis. Our data demonstrate that disruption of cellulose biosynthesis in the erl1 erl2 background does not lead to the formation of stomata clusters, indicating that cellulose biosynthesis is not a major determining factor for regulating plasmodesmatal permeability. Analysis of KOBITO1 structure suggests that it is a glycosyltransferase-like protein. KOBITO1 might be involved in a carbohydrate metabolic pathway that is essential for both cellulose biosynthesis and the regulation of plasmodesmatal permeability.

The presence of multiple introns is essential for ERECTA expression in Arabidopsis.

Gene expression in eukaryotes is often enhanced by the presence of introns. Depending on the specific gene, this enhancement can be minor or very large and occurs at both the transcriptional and post-transcriptional levels. The Arabidopsis ERECTA gene contains 27 exons encoding a receptor-like kinase that promotes cell proliferation and inhibits cell differentiation in above-ground plant organs. The expression of ERECTA very strongly depends on the presence of introns. The intronless ERECTA gene does not rescue the phenotype of erecta mutant plants and produces about 500-900 times less protein compared with the identical construct containing introns. This result is somewhat surprising as the region upstream of the ERECTA coding sequence effectively promotes the expression of extraneous genes. Here, we demonstrate that introns are essential for ERECTA mRNA accumulation and, to a lesser extent, for mRNA utilization in translation. Since mRNA produced by intronless ERECTA is degraded at the 3' end, we speculate that introns increase mRNA accumulation through increasing its stability at least in part. No individual intron is absolutely necessary for ERECTA expression, but rather multiple introns in specific locations increase ERECTA expression in an additive manner. The ability of introns to promote ERECTA expression might be linked to the process of splicing and not to a particular intron sequence.

Haploinsufficiency after successive loss of signaling reveals a role for ERECTA-family genes in Arabidopsis ovule development.

The Arabidopsis genome contains three ERECTA-family genes, ERECTA (ER), ERECTA-LIKE 1 (ERL1) and ERL2 that encode leucine-rich repeat receptor-like kinases. This gene family acts synergistically to coordinate cell proliferation and growth during above-ground organogenesis with the major player, ER, masking the loss-of-function phenotypes of the other two members. To uncover the specific developmental consequence and minimum threshold requirement for signaling, ER-family gene function was successively eliminated. We report here that ERL2 is haploinsufficient for maintaining female fertility in the absence of ER and ERL1. Ovules of the haploinsufficient er-105 erl1-2 erl2-1/+ mutant exhibit abnormal development with reduced cell proliferation in the integuments and gametophyte abortion. Our analysis indicates that progression of integument growth requires ER-family signaling in a dosage-dependent manner and that transcriptional compensation among ER-family members occurs to maintain the required signaling threshold. The specific misregulation of cyclin A genes in the er-105 erl1-2 erl2-1/+ mutant suggests that downstream targets of the ER-signaling pathway might include these core cell-cycle regulators. Finally, genetic interaction of the ER family and the WOX-family gene, PFS2, reveals their contribution to integument development through interrelated mechanisms.