ERECTA-LIKE1 Research Articles

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Multifaceted roles of the ERECTA family in plant organ morphogenesis.

Receptor-like kinases (RLKs) can participate in multiple signalling pathways and are considered one of the most critical components of the early events of intercellular signalling. As an RLK, the ERECTA family (ERf), which comprises ERECTA (ER), ERECTA-Like1 (ERL1), and ERECTA-Like2 (ERL2) in Arabidopsis, regulates multiple signalling pathways in plant growth and development. Despite its indispensability, detailed information on ERf-manipulated signalling pathways remains elusive. In this review, we attempt to summarize the essential roles of the ERf in plant organ morphogenesis, including shoot apical meristem, stem, and reproductive organ development.

The functional specificity of ERECTA-family receptors in Arabidopsis stomatal development is ensured by molecular chaperones in the endoplasmic reticulum.

Stomata are epidermal pores that control gas exchange between plants and the atmosphere. In Arabidopsis, the ERECTA family (ERECTAf) receptors, including ERECTA, ERECTA-LIKE 1 (ERL1) and ERL2, redundantly play pivotal roles in enforcing the 'one-cell-spacing' rule. Accumulating evidence has demonstrated that the functional specificities of receptors are likely associated with their differential subcellular dynamics. The endoplasmic reticulum (ER)-resident chaperone complex SDF2-ERdj3B-BiP functions in many aspects of plant development. We employed pharmacological treatments combined with cell biological and biochemical approaches to demonstrate that the abundance of ERECTA was reduced in the erdj3b-1 mutant, but the localization and dynamics of ERECTA were not noticeably affected. By contrast, the erdj3b mutation caused the retention of ERL1/ERL2 in the ER. Furthermore, we found that the function of SDF2-ERdj3B-BiP is implicated with the distinct roles of ERECTAf receptors. Our findings establish that the ERECTAf receptor-mediated signaling in stomatal development is ensured by the activities of the ER quality control system, which preferentially maintains the protein abundance of ERECTA and proper subcellular dynamics of ERL1/ERL2, prior to the receptors reaching their destination - the plasma membrane - to execute their functions.

Arabidopsis ERdj3B coordinates with ERECTA-family receptor kinases to regulate ovule development and the heat stress response.

The endoplasmic reticulum-localized DnaJ family 3B (ERdj3B), is a component of the stromal cell-derived factor 2 (SDF2)-ERdj3B-binding immunoglobulin protein (BiP) chaperone complex, which functions in protein folding, translocation, and quality control. We found that ERdj3B mutations affected integument development in the Ler ecotype but not in the Col-0 ecotype of Arabidopsis (Arabidopsis thaliana). Map-based cloning identified the ERECTA (ER) gene as a natural modifier of ERdj3B. The double mutation of ERdj3B and ER caused a major defect in the inner integument under heat stress. Additional mutation of the ER paralog ERECTA-LIKE 1 (ERL1) or ERL2 to the erdj3b er double mutant exacerbated the defective integument phenotype. The double mutation of ER and SDF2, the other component of the SDF2-ERdj3B-BiP complex, resulted in similar defects in the inner integument. Furthermore, both the protein abundance and plasma membrane partitioning of ER, ERL1, and ERL2 were markedly reduced in erdj3b plants, indicating that the SDF2-ERdj3B-BiP chaperone complex might control the translocation of ERECTA-family proteins from the endoplasmic reticulum to the plasma membrane. Our results suggest that the SDF2-ERdj3B-BiP complex functions in ovule development and the heat stress response in coordination with ERECTA-family receptor kinases.

The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate.

Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

ERECTA receptor-kinases play a key role in the appropriate timing of seed germination under changing salinity.

Appropriate timing of seed germination is crucial for the survival and propagation of plants, and for crop yield, especially in environments prone to salinity or drought. However, the exact mechanisms by which seeds perceive changes in soil conditions and integrate them to trigger germination remain elusive, especially once the seeds are non-dormant. In this study, we determined that the Arabidopsis ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2) leucine-rich-repeat receptor-like kinases regulate seed germination and its sensitivity to changes in salt and osmotic stress levels. Loss of ER alone, or in combination with ERL1 and/or ERL2, slows down the initiation of germination and its progression to completion, or arrests it altogether under saline conditions, until better conditions return. This function is maternally controlled via the tissues surrounding the embryo, with a primary role being played by the properties of the seed coat and its mucilage. These relate to both seed-coat expansion and subsequent differentiation and to salinity-dependent interactions between the mucilage, subtending seed coat layers and seed interior in the germinating seed. Salt-hypersensitive er105, er105 erl1.2, er105 erl2.1 and triple-mutant seeds also exhibit increased sensitivity to exogenous ABA during germination, and under salinity show an enhanced up-regulation of the germination repressors and inducers of dormancy ABA-insensitive-3, ABA-insensitive-5, DELLA-encoding RGL2, and Delay-Of-Germination-1. These findings reveal a novel role of the ERECTA receptor-kinases in the sensing of conditions at the seed surface and the integration of developmental, dormancy and stress signalling pathways in seeds. They also open novel avenues for the genetic improvement of plant adaptation to changing drought and salinity patterns.

Auxin and Gibberellins Are Required for the Receptor-Like Kinase ERECTA Regulated Hypocotyl Elongation in Shade Avoidance in Arabidopsis.

Plants use shade avoidance strategy to escape the canopy shade when grown under natural conditions. Previous studies showed that the Arabidopsis receptor-like kinase ERECTA (ER) is involved in shade avoidance syndrome. However, the mechanisms of ER in modulating SAR by promoting hypocotyl elongation are unknown yet. Here, we report that ER regulated hypocotyl elongation in shade avoidance requires auxin and gibberellins (GAs). The T-DNA insertional ER mutant er-3 shows a less hypocotyl length than that in Col-0 wild type. Promoter::GUS staining analysis shows that ER and its paralogous genes ERECTA-LIKE1 (ERL1) and ERECTA-LIKE2 (ERL2) are differentially expressed in the seedlings, of which only ER is most obviously upregulated in the hypocotyl by shade treatment. Exogenous feeding assay by using media-application with vertical-grown of Arabidopsis seedlings showed that the hypocotyl length of er-3 is partially promoted by indol-3-acetic acid (IAA), while it is relatively insensitive of er-3 to various concentrations of IAA than that of Col-0. Hypocotyl elongation of er-3 is promoted similar to that of Col-0 by high temperature in the white light condition, but the elongation was not significantly affected by the treatment of the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). Exogenous GA3 increased the hypocotyl elongation of both er-3 and the wild type in the shade condition, and the GA3 biosynthesis inhibitor paclobutrazol (PAC) severely inhibits the hypocotyl elongation of Col-0 and er-3. Further analysis showed that auxin biosynthesis inhibitors yucasin and L-kynurenine remarkably inhibited the hypocotyl elongation of er-3 while yucasin shows a more severe inhibition to er-3 than Col-0. Relative expression of genes regulating auxin homeostasis and signaling, and GA homeostasis is less in er-3 than that in Col-0. Furthermore, genetic evidences show that ER regulated hypocotyl elongation is dependent of PHYTOCHROME B (PHYB). Overall, we propose that ER regulated shade avoidance by promoting hypocotyl elongation is PHYB-dependent and requires auxin and GAs.

ERECTA Regulates Cell Elongation by Activating Auxin Biosynthesis in Arabidopsis thaliana.

The ERECTA family genes, ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2), encode leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Knocking out these three genes can cause severe phenotypes, which indicates that they play significant roles in plant growth and development. However, the molecular mechanism within remains unclear. Here we show that the short hypocotyl phenotypes of er erl1 erl2 mutants are mainly due to the defects of cell elongation rather than the cell division. In contrast, in the ERECTA overexpression transgenic plants, the hypocotyl length is increased with elongated cells. Moreover, we show that the er erl1 erl2 triple mutant contains a low level of auxin, and the expression levels of the key auxin biosynthesis genes are significantly reduced. Consistent with this observation, increasing exogenous or endogenous auxin levels could partially rescue the cell elongation defects of the er erl1 erl2 triple mutant. Therefore, our results provide a molecular basis for auxin mediated ERECTA control of the hypocotyl length in Arabidopsis thaliana.

Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling

Development of stomata, valves on the plant epidermis for optimal gas exchange and water control, is fine-tuned by multiple signaling peptides with unique, overlapping, or antagonistic activities. EPIDERMAL PATTERNING FACTOR1 (EPF1) is a founding member of the secreted peptide ligands enforcing stomatal patterning. Yet, its exact role remains unclear. Here, we report that EPF1 and its primary receptor ERECTA-LIKE1 (ERL1) target MUTE, a transcription factor specifying the proliferation-to-differentiation switch within the stomatal cell lineages. In turn, MUTE directly induces ERL1. The absolute co-expression of ERL1 and MUTE, with the co-presence of EPF1, triggers autocrine inhibition of stomatal fate. During normal stomatal development, this autocrine inhibition prevents extra symmetric divisions of stomatal precursors likely owing to excessive MUTE activity. Our study reveals the unexpected role of self-inhibition as a mechanism for ensuring proper stomatal development and suggests an intricate signal buffering mechanism underlying plant tissue patterning.

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.

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.

Direct interaction of ligand–receptor pairs specifying stomatal patterning

Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2-ERECTA and EPF1-ERL1 as ligand-receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

A view from the top: new ligands controlling stomatal development in Arabidopsis

A view from the top: new ligands controlling stomatal development in Arabidopsis

Epidermal Cell Density is Autoregulated via a Secretory Peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis Leaves

Regulation of the number of cells is critical for development of multicellular organisms. During plant epidermal development, a protodermal cell first makes a fate decision of whether or not to be the meristemoid mother cell (MMC), which undergoes asymmetric cell division forming a meristemoid and its sister cell. The MMC-derived lineage produces all stomatal guard cells and a large proportion of non-guard cells. We demonstrate that a small secretory peptide, EPIDERMAL PATTERING FACTOR 2 (EPF2), is produced by the MMC and its early descendants, and negatively regulates the density of guard and non-guard epidermal cells. Our results suggest that EPF2 inhibits cells from adopting the MMC fate in a non-cell-autonomous manner, thus limiting the number of MMCs. This feedback loop is critical for regulation of epidermal cell density. The amino acid sequence of EPF2 resembles that of EPF1, which is known to control stomatal positioning. Over-expression of EPF1 also inhibits stomatal development, but EPF1 can act only on a later developmental process than EPF2. Overexpression and promoter swapping experiments suggested that the protein functions of EPF1 and EPF2, rather than the expression patterns of the genes, are responsible for the specific functions. Although targets of EPF1 and EPF2 are different, both EPF1 and EPF2 require common putative receptor components TOO MANY MOUTHS (TMM), ERECTA (ER), ERECTA LIKE 1 (ERL1) and ERL2 in order to function.

Regulation of Arabidopsis Early Anther Development by the Mitogen-Activated Protein Kinases, MPK3 and MPK6, and the ERECTA and Related Receptor-Like Kinases

Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/- and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/- mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/- mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.

Haploinsufficiency after successive loss of signaling reveals a role forERECTA-family genes inArabidopsisovule 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.