Plants have evolved a signalling pathway in which, when one root senses local nitrogen (N) deficiency, nitrate uptake by other roots is enhanced in a complementary manner. This long-range communication, known as systemic N-demand signalling, is triggered when the root-to-shoot mobile signal, C-TERMINALLY ENCODED PEPTIDE (CEP), which is induced in roots under N starvation, is perceived by CEP RECEPTOR 1 (CEPR1) expressed in the leaf phloem. However, the molecular components required for CEP-dependent CEPR1 activation remain unknown. Here we identified a leucine-rich repeat receptor kinase that interacts with CEPR1 in a CEP-dependent manner, which we named CEP RECEPTOR INTERACTOR (CERI). CERI belongs to the last functionally uncharacterized clade within the Arabidopsis leucine-rich repeat receptor kinase subgroup II. Loss of CERI impairs systemic N-demand signalling but does not affect CEPR1-mediated regulation of root system architecture. CERI functions as a co-receptor that confers signalling specificity on CEPR1 by selectively mediating systemic N-demand signalling. This study identifies a LRR-RK subfamily II receptor kinase as a co-receptor for CEPR1, assigning a function to the last unknown clade of this receptor family and revealing a key regulator of systemic nitrogen-demand signalling in plants.

Meristem activity is controlled by the CLAVATA (CLV) signaling pathway, which involves a suite of leucine-rich receptor (LRR) receptors, receptor-like proteins, and CLV-EMBRYO SURROUNDING REGION (CLE) peptide ligands. FASCIATED EAR 3 (FEA3) is a leucine-rich receptor (LRR) receptor-like protein important for meristem maintenance in maize and acts independently of canonical CLV receptors. To identify FEA3's interaction network, we used TurboID-based proximity labeling in Zea mays meristems and identified a putative co-receptor, BARELY ANY MERISTEM 1D (BAM1D). BAM1D and FEA3 proximity labeling proteomes shared over 40 proteins, including many signaling proteins, suggesting they feed into a common signaling pathway. fea3 was epistatic to bam1d in the control of inflorescence meristem (IM) size, supporting the idea that FEA3 and BAM1D interact physically. However, fea3 and bam1d act antagonistically because fea3 mutants had larger IMs, whereas bam1d mutants produced smaller IMs. This study demonstrates how in vivo TurboID-based proximity labeling clarifies complex genetic interactions between CLV receptors and expands our knowledge of downstream signaling components of CLV signaling pathways, which are largely uncharacterized. Our findings support the notion that multiple, partially overlapping CLV receptor complexes coordinately control meristem maintenance.

In response to drought stress, land plants close their stomata to minimize transpiration. This action precedes a gradual accumulation of the stress hormone abscisic acid (ABA) that enhances plant drought tolerance. However, the molecular mechanisms that cause the time lag between the onset of stomatal closure and ABA accumulation and coordinate these two phases remain unexplained. Here, we found that Arabidopsis thaliana loss-of-function CLAVATA3/ENDOSPERM SURROUNDING REGION 5 (CLE5) mutants are less tolerant to drought. The CLE5 dodecapeptide (CLE5p) acts as a local signal to induce stomatal closure by binding to the LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASE (LRR-RLK) receptor complex, BARELY ANY MERISTEM 1 (BAM1)–GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1), in guard cells. The BAM1–GHR1–CLE5p module directly phosphorylates two SNF1-related protein kinases, OPEN STOMATA1 (SRK2E) and SRK2D, the central regulators of drought responses in plants, to regulate stomatal movement and drought-responsive gene expression without stimulating ABA biosynthesis or ROS accumulation. Our findings mark a critical step in understanding how plants promptly counteract environmental stresses. The CLEp–LRR-RLK signalling components are highly conserved across plant phyla, suggesting that peptide-mediated rapid stomatal closure is a widespread survival strategy and can be exploited to generate drought-resistant crops. Shimotohno et al. demonstrated a BAM1–GHR1–CLE5p/SnRK2 signal transduction module that regulates primary drought responses without stimulating ABA biosynthesis in Arabidopsis.

Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the Arabidopsis apoplastic fluid. The method involves using Arabidopsis-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry-based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast. Key features • Targets Arabidopsis thaliana secreted protease but may be applicable to other plant species and intracellular proteases if protease-enriched samples are available. • The protocol involves an in-gel peptide cleavage assay of native two-dimensional gels diced with SAINOME plates, using a fluorescence-quenching substrate. • Facilitates the efficient identification of proteases with the desired activity from the entire sample, without restricting the analysis to a specific class of proteases.

In the accumulation response, chloroplasts move toward weak blue light (BL) to maximize photosynthetic efficiency; in the avoidance response, they move away from strong BL to reduce photodamage. The BL receptor kinase phototropin (phot) mediates these chloroplast relocation responses, and the chloroplast relocation response requires phot kinase activity. Upon receiving BL, phot undergoes autophosphorylation; however, the molecular mechanisms that regulate chloroplast relocation through phot autophosphorylation remain unclear. In this study, we conducted biochemical experiments using phot in the liverwort Marchantia polymorpha and revealed that phot employs cis-autophosphorylation under weak BL and both cis- and trans-autophosphorylation under strong BL. Inhibiting trans-autophosphorylation reduced phot autophosphorylation and suppressed the avoidance response, but not the accumulation response. These findings suggest that phot employs two modes of autophosphorylation to alternate between the accumulation and avoidance responses in plants.

Various secreted peptides, including peptide hormones, are present in the apoplast, but their biochemical characterization remains a challenge due to their low abundance, difficulty in extraction, and interference from numerous secondary metabolites. Here, we describe a simple and straightforward protocol for the extraction of apoplastic peptides with a high purity. This protocol takes advantage of the fact that apoplastic peptides diffuse and accumulate in the culture medium when Arabidopsis seedlings are subjected to whole-plant submerged culture. The peptides in the culture medium are efficiently recovered by o-chlorophenol extraction followed by acetone precipitation. The recovered peptides can be subjected to nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) without any additional clean-up. This procedure enables the structural elucidation of mature peptide hormones in the apoplast with the use of Arabidopsis plants that overexpress peptide hormone genes.

Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but are impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions. As sessile organisms, plants grow under continuously fluctuating nitrogen (N) nutrient environments. Here, the authors uncovered that root TGA1/4, as key transcription factors, regulate the expression of nitrate uptake genes by binding to shoot-derived transcriptional cofactors that reflect shoot N demand.

Binding assays provide ultimate proof that a particular peptide and receptor kinase (RK) do indeed function as a ligand-receptor pair. Among available binding assays, proximity-induced photoaffinity labeling is superior for confirming direct contact between the peptide ligand and the receptor. Our binding assay employs covalent photoaffinity labeling followed by immunoprecipitation to specifically evaluate the ligand binding activity of the target RKs. Here, we describe a protocol for the synthesis of photoactivatable peptide ligands and the UV-induced formation of covalent bonds between photoaffinity ligands and RKs.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana . These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

Small interference RNA (siRNA) selectively accumulates and acts in RNA interference (RNAi). Although the components involved in siRNA production have long been the focus of studies to elucidate RNAi processes, the mechanism(s) for selectivity of siRNA (or RNAi effectivity) remains unclear. In a novel approach, we developed a progressive deep learning (DL) framework integrating Transformer and convolutional neural networks to predict the sequences of selectively accumulated siRNAs across various land plant species. These approaches achieved high-accuracy prediction of selectively accumulated 21-nt siRNAs and further identified their key signals, which are positionally and linguistically flexible sequences surrounding the target siRNA. We experimentally validated the contribution of these flexible key signal sequences to siRNA accumulation selectivity using virus-induced gene silencing (VIGS) in Nicotiana benthamiana, and identified RNA-binding proteins that directly recognize the key signal sequences to act for selective siRNA accumulation. These insights provide a novel framework for investigating RNAi mechanisms in plants.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

Arabinogalactan proteins (AGPs) are a plant-specific family of extracellular proteoglycans characterized by large and complex galactose-rich polysaccharide chains. Functional elucidation of AGPs, however, has been hindered by the high degree of redundancy of AGP genes. To uncover as yet unexplored roles of AGPs in Arabidopsis, a mutant of Hyp O-galactosyltransferase (HPGT), a critical enzyme that catalyzes the common initial step of Hyp-linked arabinogalactan chain biosynthesis, was used. Here we show, using the hpgt1,2,3 triple mutant, that a reduction in functional AGPs leads to a stomatal patterning defect in which two or more stomata are clustered together. This defect is attributed to increased and dysregulated symplastic transport following changes in plasmodesmata structure, such that highly permeable complex branched plasmodesmata with cavities in branching parts increased in the mutant. We also found that the hpgt1,2,3 mutation causes a reduction of cellulose in the cell wall and accumulation of pectin, which controls cell wall porosity. Our results highlight the importance of AGPs in the correct biogenesis of plasmodesmata, possibly acting through the regulation of cell wall properties surrounding the plasmodesmata.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

Deciding whether to grow or to divert energy to stress responses is a major physiological trade-off for plants surviving in fluctuating environments. We show that three leucine-rich repeat receptor kinases (LRR-RKs) act as direct ligand-perceiving receptors for PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides and mediate switching between two opposing pathways. By contrast to known LRR-RKs, which activate signaling upon ligand binding, PSY receptors (PSYRs) activate the expression of various genes encoding stress response transcription factors upon depletion of the ligands. Loss of PSYRs results in defects in plant tolerance to both biotic and abiotic stresses. This ligand-deprivation-dependent activation system potentially enables plants to exert tuned regulation of stress responses in the tissues proximal to metabolically dysfunctional damaged sites where ligand production is impaired.

2 O 等)の発生，土壌の酸性化，生物多様性の 減少などの環境問題が地球規模の広がりを見せている (4) ． 窒素肥料の生産には大量の化石燃料が必要であることか らも，窒素肥料使用量の削減が国際社会の喫緊の課題と なっている (5) ．この課題解決に向けた植物科学的アプ ローチとして，窒素肥料の量を減らしても生産性が低下 しない農作物の開発が期待されているが，そのためには 植物の低窒素環境における生存戦略 窒素不足に適応する巧妙な仕組み

The nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation 1 , 2 . However, the critical enzymes required for the post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating Ser501 of NRT2.1, a residue that functions as a negative phospho-switch in Arabidopsis 2 . CEPH is predominantly expressed in epidermal and cortex cells in roots and is upregulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signalling from shoots 3 , 4 . The loss of CEPH leads to marked decreases in high-affinity nitrate uptake, tissue nitrate content and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in the N-starvation-dependent activation of NRT2.1 and provide molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilases including SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis. Introduction Complementing the activity of the classical phytohormones, peptide hormones and growth factors are now recognized as an important class of signaling molecules for long-range signaling and for cell-to-cell communication over short distances, respectively (Oh et al., 2018; Stührwohldt and Schaller, 2019). In Arabidopsis thaliana (hereafter ‘Arabidopsis’), there are more than 1000 genes potentially encoding signaling peptides, apparently involved in all aspects of plant growth and development (Lease and Walker, 2006; Ghorbani et al., 2015; Tavormina et al., 2015). There has been remarkable progress in recent years with respect to the characterization of peptide perception and signal transduction mechanisms (Song et al., 2017; He et al., 2018). The biogenesis of these signaling molecules, on the other hand, is still poorly understood. This is particularly true for the large group of signaling peptides that depend on a series of post-translational modifications (PTMs) for maturation and activation (Matsubayashi, 2014; Stührwohldt and Schaller, 2019). Proteolytic processing is required for all post-translationally modified signaling peptides to release the peptide entity from its precursor. Additional PTMs may include tyrosine sulfation, proline hydroxylation, and arabinosylation of the hydroxyproline residue (Matsubayashi, 2014; Stührwohldt and Schaller, 2019). Tyrosine sulfation is performed by a single tyrosylprotein sulfotransferase (TPST) that is membrane-anchored in the cis-Golgi (Komori et al., 2009). TPST requires aspartate on the amino side of tyrosin for substrate recognition (Komori et al., 2009). Tyrosine sulfation is a critical maturation step, as sulfated peptides usually depend on this modification for full activity (Stührwohldt and Schaller, 2019). Proline hydroxylation is catalyzed by membrane-anchored prolyl-4-hydroxylases (P4Hs) localized in ER and Golgi compartments. There are 13 P4Hs in Arabidopsis, some of which were shown to be required for the hydroxylation of extensin and possibly other hydroxyprolin (Hyp)-rich glycoproteins of the cell wall (Velasquez et al., 2015). Which of the P4Hs act on signaling peptides, and whether or not they differ in preference for proline in a certain sequence context is still unclear. Proline hydroxylation is a prerequisite for subsequent glycosylation. As the first in a series of glycosylation steps, L-arabinose is transferred to the 4-hydroxyl by Golgi-resident Hydroxyproline O-arabinosyltransferase (HPAT). HPAT is encoded by three genes in Arabidopsis that are at least partially functionally redundant (Ogawa-Ohnishi et al., 2013; MacAlister et al., 2016). To what extent differences in substrate specificity of HPATs may contribute to the selection of certain Hyp residues for glycosylation remains to be seen. The identification of precursor processing proteases lags behind the other PTM enzymes. It was and is still hampered by the large number of possible candidates (907 peptidases are listed in the MEROPS database (release 12.0) for Arabidopsis Rawlings et al., 2016), by their generally low expression levels, by functional redundancy, and by the lack of a conserved processing site. As compared to tyrosine sulfation, proline hydroxylation and Hyp arabinosylation which occur at Asp-Tyr, Pro, and Hyp residues, respectively, and in contrast to animal systems, where peptide hormones are typically flanked by pairs of basic residues, there is no consensus motif that would mark the cleavage sites for processing of peptide precursors in plants (Rawlings et al., 2016; Stührwohldt and Schaller, 2019). We may thus expect that many different proteases with different specificities for cleavage site selection are required for the processing of the many different precursors. Consistent with this notion, precursor processing enzymes have been identified in different classes of proteases, including a metacaspase and a papain-like enzyme among the cysteine peptidases (Wrzaczek et al., 2015; Ziemann et al., 2018; Hander et al., 2019), a carboxypeptidase in the class of the zink-dependent metallo peptidases (Casamitjana-Martínez et al., 2003), and several subtilases (SBTs) among the serine peptidases (Srivastava et al., 2009; Engineer et al., 2014; Ghorbani et al., 2016; Schardon et al., 2016; Stegmann et al., 2017; Beloshistov et al., 2018; Doll et al., 2020; Reichardt et al., 2020). SBTs thus seem to play a more general role in peptide hormone maturation (Schaller et al., 2018). SBTs constitute a large family of mostly extracellular proteases including e.g. 56 members in Arabidopsis (Rautengarten et al., 2005), 86 in tomato (Reichardt et al., 2018) and 97 in grapevine (Figueiredo et al., 2016). Expansion of the SBT family in plants involved both whole genome and tandem gene duplications with differential neo- and sub-functionalization resulting in many taxon-specific clades (Taylor and Qiu, 2017; Reichardt et al., 2018). AtSBT6.1 stands out in this diverse family for several reasons. First, unlike most other plant SBTs that are soluble proteins targeted to the cell wall (Schaller et al., 2018), AtSBT6.1 is a membrane protein, anchored by a C-terminal membrane-spanning helix to the Golgi and possibly the plasma membrane (Liu et al., 2007a; Ghorbani et al., 2016). Second, AtSBT6.1 is one of only two Arabidopsis SBTs that originated before the divergence of Metazoa and Viridiplantae, and is functionally conserved between animals and plants (Taylor and Qiu, 2017; Schaller et al., 2018). Like Site-1-Protease (S1P), its orthologue in humans, AtSBT6.1 (alias AtS1P) cleaves membrane-anchored bZIP transcription factors in the Golgi to facilitate the translocation of their cytoplasmic domain to the nucleus for the induction of ER stress-response genes (Liu et al., 2007b; Liu et al., 2007a). Furthermore, the cleavage site preference of AtSBT6.1 for the amino acid motives R-(R/K)-X-L or R-(R/K)-L-X (Srivastava et al., 2009; Ghorbani et al., 2016) is almost identical with the substrate specificity reported for human S1P (R-X-(L/V/I)-X; Seidah, 2013). With its predominant Golgi localization (Liu et al., 2007a; Parsons et al., 2012), AtSBT6.1 seems predestined to act in concert with TPST, P4Hs and HPATs in the biogenesis of post-translationally modified signaling peptides. Supporting this notion, potential AtSBT6.1/S1P cleavage sites can be found in many peptide precursors including members of the Rapid Alkalinization Factor (RALF), phytosulfokine (PSK), Clavata3/Embryo Surrounding Region (CLE) and CLE-Like (CLEL) families. The latter is also known as Golven (GLV) or Root Meristem Growth Factor (RGF) family and comprises 11 precursor-derived peptides of 13 to 18 amino acids carrying two additional post-translational modifications, i.e. tyrosine sulfation and hydroxylation of the ultimate proline residue (Matsuzaki et al., 2010; Meng et al., 2012; Whitford et al., 2012). We refer to them here as CLEL, because not all family members are involved in root gravitropism (causing the GLV phenotype), or regulating the activity of the root apical meristem (as the name RGF would suggest). However, processing by AtSBT6.1 has so far only been shown for RALF23 (Srivastava et al., 2009; Stegmann et al., 2017), and in the particularly interesting case of CLEL6 (GLV1/RGF6) (Ghorbani et al., 2016). AtSBT6.1 was identified as a factor required for CLEL6 function in a screen for sbt mutants suppressing the CLEL6-overexpression phenotype (agravitropic root growth and increased hypocotyl elongation) (Ghorbani et al., 2016). The protease was shown to cleave the CLEL6 precursor at two canonical AtSBT6.1/S1P cleavage sites (R-R-L-R, R-R-A-L), and both cleavage sites turned out to be relevant for CLEL6 function, the second one even essential. The data indicate that AtSBT6.1 activity is required for the formation of the bioactive CLEL6 peptide (Ghorbani et al., 2016). Surprisingly however, AtSBT6.1 cleavage sites are located in the variable part of the CLEL6 and other peptide precursors, considerably upstream of the mature peptide sequence. AtSBT6.1 activity is thus not sufficient and additional unknown protease(s) are required for peptide maturation. Completely unresolved is the question when and where the processing of peptide precursors takes place, particularly in relation to the other PTMs. While the Golgi is an obvious possibility for processing by AtSBT6.1, the enzyme has also been reported at the cell surface (Ghorbani et al., 2016) suggesting apoplastic processing of the fully modified precursor as an alternative possibility. This has implicitly been assumed for cell wall-localized SBTs. However, as secretory enzymes they are co-targeted with their potential peptide precursor substrates providing ample opportunity for processing en route, in any compartment of the secretory pathway. These are the questions that are addressed here for the CLEL6 and CLEL9 peptide precursors. Results SBT activity is required for the maturation of CLEL6 and CLEL9 peptides In order to confirm the involvement of SBTs in the maturation of CLEL6 (GLV1/RGF6), we used the inhibitor-based loss-of-function approach that was previously employed to demonstrate a role for redundant SBTs in the maturation of IDA (Inflorescence Deficient in Abscission) resulting in the shedding of Arabidopsis flower organs after pollination (Schardon et al., 2016; Stührwohldt et al., 2017; Stührwohldt et al., 2018). CLEL9 (GLV2/RGF9) was included in the analysis because it acts redundantly with CLEL6 in the regulation of gravitropic responses (Whitford et al., 2012), and because it resembles CLEL6 with respect to predicted processing sites (Figure 1—figure supplement 1A). The SBT-specific Extracellular Proteinase Inhibitors (EPIs) 1a and 10 from Phytophthora infestans were expressed in transgenic Arabidopsis plants under control of the CLEL6 or CLEL9 promoters (Figure 1—figure supplement 1B). Inhibition of SBTs by EPIs in tissues where CLEL6 and CLEL9 are expressed is expected to phenocopy the CLEL6/9 loss-of-function phenotype if SBT activity is required for precursor processing and peptide maturation. Seedlings expressing EPI1a under the control of either the CLEL6 or the CLEL9 promoter were impaired in the gravitropic response of the hypocotyl (Figure 1A,C). Likewise, hypocotyl gravitropism was inhibited also by the expression of EPI10 controlled by either one of the two CLEL promoters (Figure 1—figure supplement 2). The same phenotype had been observed when CLEL6 or 9 were silenced in transgenic plants by artificial micro RNAs (Whitford et al., 2012), suggesting that SBT activity is required for CLEL6 and 9 function. However, in contrast to CLEL6 or 9-silenced plants (Whitford et al., 2012), the gravitropic response of roots was not affected in our EPI-expressing transgenics (Figure 1—figure supplement 3). This observation is consistent with the fact that the CLEL6 and 9 promoters are active in the hypocotyl where they drive the expression of EPI inhibitors in epidermis and cortex (Whitford et al., 2012), but not in any part of the primary root (Fernandez et al., 2013). Figure 1 with 3 supplements see all Download asset Open asset Reduced gravicurvature of PCLEL6:EPI1a, PCLEL9:EPI1a and tpst-1 seedlings is rescued by addition of mature CLEL6 or CLEL9 peptides. Gravicurvature of (A) three independent PCLEL6:EPI1a and (C) PCLEL9:EPI1a lines (gray bars; promoter constructs are shown in Figure 1—figure supplement 1) is significantly reduced in comparison to the wild type (white bars). The same effect was observed when the EPI10 inhibitor was expressed under control of the CLEL6 or CLEL9 promoter (Figure 1—figure supplement 2). Gravicurvature of PCLEL6:EPI1a and PCLEL9:EPI1a lines is restored to wild-type levels by application of (B) 10 nM CLEL6 or (D) 300 nM CLEL9 (hatched bars). (E) tpst-1 gravicurvature in comparison to the wild-type control. (F) Gravicurvature of tpst-1 seedlings treated (hatched bars) with CLEL6 (10 nM) or CLEL9 (300 nM) as compared to the untreated tpst-1 control (gray bars). Seedlings were grown for five days in the dark on ½ MS medium with peptides added as indicated. Plates were rotated 90° and gravicurvature was assessed after two days as the angle of the hypocotyl with the horizontal. Panel A was modified from Stührwohldt et al. (2017). Data are shown for one representative of at least two independent experiments as the mean ± SE (n ≥ 15). *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively (two-tailed t test). The gravitropic response of roots was not affected in transgenic plants expressing the EPI inhibitors under control of CLEL promoters (Figure 1—figure supplement 3). Figure 1—source data 1 Source data for hypocotyl gravitropic responses shown in Figure 1 and Figure 1—figure supplement 2. https://cdn.elifesciences.org/articles/55580/elife-55580-fig1-data1-v1.xlsx Download elife-55580-fig1-data1-v1.xlsx When plants expressing the EPI1a inhibitor were supplied with synthetic CLEL6 or CLEL9 peptides, gravitropism of the hypocotyl was restored to wild-type level (Figure 1B,D). Likewise, we observed an impaired gravitropic response in mutants defective in tyrosylprotein sulfotransferase (TPST), and the defect of the tpst-1 mutant also was alleviated by application of the sulfated CLEL6 or CLEL9 peptides (Figure 1E,F). The data indicate that the peptides act downstream of SBT (and TPST) activity, consistent with a role for SBTs (and TPST) in peptide maturation (Figure 1B,D–F). The data are fully consistent with findings of Ghorbani et al. (2016), who reported that the activity of SBT6.1 is required for CLEL6 function, and they further indicate that SBTs are required also for the activation of CLEL9. The CLEL6 precursor comprises two potential S1P (SBT6.1) cleavage sites, RRLR and RRAL (Figure 2A), and the second site is necessary for CLEL6 function (Ghorbani et al., 2016). However, cleavage by SBT6.1 is not sufficient for CLEL6 formation, since both sites are located considerably upstream of the mature peptide sequence. We thus refer to the cleavage by SBT6.1 as a necessary pre-processing step that precedes peptide activation (Stührwohldt and Schaller, 2019). Additional protease(s) are needed to mark the N-terminus and release the fully processed CLEL6 peptide. Whether the final processing for peptide activation also is mediated by SBTs, is still unclear at this time. Also unclear are the subcellular sites of pre-processing and peptide activation, and the sequence of post-translational modification events. These questions were addressed in the following. Figure 2 Download asset Open asset Pre-processing of CLEL6 by SBT6.1 in the cis-Golgi is required for peptide maturation and secretion. (A) Sequence of the CLEL6 precursor without the signal peptide. Proteolytic processing sites and other post-translational modifications are indicated, mature CLEL6 peptide sequence underlined. Colored lines and asterisks represent the size of the processed forms of CLEL6 observed in panels C, E, and F. (B) Schematic representation of expression constructs used for subcellular localization studies. (C) Processing of the ER-restrained CLEL6 (KDEL) construct with (+) or without (-) BFA treatment, analyzed by anti-GFP immunoblotting. Different processed forms of the precursor are marked by colored asterisks as shown in panel A. (E, F) Immunoblot analysis of ER-restrained (KDEL) and Golgi-localized (XylT) precursor processing compared to the secreted form (Sec) and the precursor lacking the two SBT6.1 cleavage sites (Secm). Fully and partially processed precursors are indicated by the colored asterisks as defined in panel A. (D, G–I) Co-localization of the different fusion proteins with ER (VMA12-mRFP) and Golgi (ManI-mCherry and ST-mCherry) markers analyzed by fluorescence microscopy. Pictures show an overlay of the green (500–550 nm) and red (610–670 nm) fluorescence channels. The dotted areas are shown in higher magnification in the insets. Scale bars represent 5 µm. Pre-processing by SBT6.1 in an early Golgi compartment is required for secretion SBT6.1 is known to be active in the Golgi, where it is required for the processing and activation of membrane-anchored transcription factors and of PMEs (Liu et al., 2007a; Liu et al., 2007b; Wolf et al., 2009; Sénéchal et al., 2014), and it was reported also in the cell wall, where it was detected in complex with the Serpin1 inhibitor (Ghorbani et al., 2016). To address the question whether pre-processing by SBT6.1 occurs within the secretory pathway or extracellularly, we transiently expressed the CLEL6 precursor fused to sfGFP in N. benthamiana. The sfGFP tag was linked to the N-terminus of the precursor, just downstream of the signal peptide (construct named ‘Sec’ in Figure 2B). GFP fluorescence was detected only in the apoplast (Figure 2D). On an anti-GFP immunoblot a single band was detected corresponding in size to GFP with part of the precursor up to the first SBT6.1 cleavage site (Figure 2E, red asterisk). Processing at this site is thus efficient when the precursor is allowed to pass through the secretory pathway. When the precursor was equipped with a C-terminal KDEL-motif for ER retention (construct named ‘KDEL’ in Figure 2B), processing was incomplete (Figure 2E). In addition to the apoplast, the fluorescence signal was now detected also in the ER and Golgi, as indicated by co-expression of ER (Vma12-mRFP) or Golgi (ManI-mCherry and ST-mCherry for early and late Golgi, respectively) markers (Figure 2G). The signal in the ER and early Golgi compartments results from the unprocessed precursor, while the presence of extracellular GFP indicates that some of the precursor was processed to separate GFP from the ER retention signal. Partial processing also is apparent on the western blot, where two additional bands were observed (Figure 2E), which we interpret as the precursor processed at the second SBT6.1 site (green asterisk), and the unprocessed precursor (black asterisk), respectively. ER retention by the KDEL-motif is mediated by Golgi-resident K/HDEL-receptors, which effect retrograde transport of soluble ER proteins from the Golgi back to the ER (Pelham, 1988; Phillipson et al., 2001; Silva-Alvim et al., 2018). Cleavage by SBT6.1 may thus occur either in the ER or in the Golgi. However, processing was abolished when anterograde ER-to-Golgi vesicle transport was inhibited by addition of brefeldin A (Nebenführ et al., 2002; Figure 2C). These observations indicate that exit from the ER is required for cleavage by SBT6.1, and we conclude that SBT6.1 acts in the Golgi, likely in an early Golgi compartment. This conclusion was confirmed by fusing the CLEL6 precursor to the N-terminal membrane anchor of ß−1,2-xylosyltransferase (XylT), which is sufficient to target reporter proteins to the medial Golgi (Pagny et al., 2003; Figure 2H). The same three cleavage products were observed as for the KDEL-tagged precursor at somewhat different ratios (Figure 2E) indicating that cleavage by SBT6.1 occurs before the precursor reaches the trans Golgi network (TGN). To assess the relevance of cleavage by SBT6.1 for processing and secretion, we masked both cleavage sites (Figure 2A; RRLR and RRRAL) by alanine substitutions as described by Ghorbani et al. (2016) and analyzed the effect on the processing pattern of the transiently expressed precursor (compare constructs ‘Sec’ and ‘Secm’ in Figure 2F). The central band corresponding to the second cleavage site was lost for the Ala-substituted precursor confirming that SBT6.1 is responsible and necessary for this cleavage event. This may not be the case for the first cleavage event, as the corresponding band was still observed for the Ala-substituted precursor, suggesting that another protease may jump in when cleavage by SBT6.1 is prevented, or a different protease cleaves the mutant cleavage site. Interestingly, comparing Figure 2D and I, we observed that secretion of the Ala-substituted precursor is reduced compared to the wild-type. For the Ala-substituted precursor, the GFP fluorescence signal was observed in both ER and Golgi in addition to the apoplastic space suggesting that cleavage by SBT6.1 may facilitate continued passage along the secretory pathway and, hence, additional post-translational modifications in post-Golgi compartments. This observation may explain why pre-processing by SBT6.1 is required for CLEL6 function in vivo (Ghorbani et al., 2016), despite the fact that this cleavage event does not produce the mature peptide. The cleavage for final activation occurs in a post-Golgi compartment by aspartate-dependent subtilase SBT3.8 After pre-processing of CLEL6 by SBT6.1 in the Golgi, additional processing at the N-terminus is required for maturation and activation. To localize this processing event subcellularly, we used an N-terminally sfGFP-tagged deletion construct of the CLEL6 precursor (Δ-Sec) lacking both SBT6.1 processing sites (Figure 3A). Again, we analyzed a secreted version (Δ-Sec), one that was equipped with a C-terminal KDEL-motif for ER retention (Δ-KDEL) and one that was anchored to the Golgi membrane (Δ-XylT, Figure 3B). On an anti-GFP immunoblot a single band was detected for Δ-Sec corresponding in size to the precursor processed at the N-terminal maturation site (Figure 3C, blue asterisk). Interestingly, for Δ-Sec some of the GFP signal was observed in the cell wall, in addition to the ER and a weak signal in the late Golgi (ST-mCherry marker in Figure 3D). Secretion of this construct, like that of Secm (Figure 2I), is thus reduced compared to wild-type Sec (Figure 2D), suggesting that the propeptide, in addition to propeptide cleavage by SBT6.1 (Figure 2I), may contribute to efficient passage through the secretory pathway. Figure 3 with 1 supplement see all Download asset Open asset N-terminal maturation of CLEL6 occurs in a post-Golgi compartment (the subcellular localization of CLEL9 maturation is analyzed in Figure 3—figure supplement 1). (A) Sequence and post-translational modification sites of Δ-CLEL6 constructs lacking the prodomain region encompassed by the two SBT6.1 cleavage sites; mature CLEL6 peptide sequence underlined. Black and blue lines and asterisks were included to represent the unprocessed and processed forms of the precursor, respectively. (B) Schematic representation of expression constructs used to localize the subcellular compartment of CLEL6 maturation. (C) Processing of the secreted (Δ-Sec), ER-restrained (Δ-KDEL) and Golgi-localized (Δ-XylT) constructs analyzed by anti-GFP immunoblotting. Unprocessed and processed forms of the precursor are indicated by the black and blue asterisks, respectively. (D–F) Co-localization of the different fusion proteins with ER (VMA12-mRFP) and Golgi (ManI-mCherry and ST-mCherry) markers analyzed by fluorescence microscopy. Pictures show an overlay of the green (500–550 nm) and red (610–670 nm) fluorescence channels. The dotted areas are shown in higher magnification in the insets. The white arrow marks apoplastic localization; scale bars represent 5 µm. In contrast to Δ-Sec, the unprocessed precursors were observed for both Δ-KDEL and Δ-XylT as single larger bands on the immunoblot (Figure 3C, black asterisk). The GFP signal for the C-terminally KDEL-tagged deletion (Δ) construct was found exclusively in the ER (Figure 3E). Retention of the N-terminal GFP tag in the ER confirmed that processing did not occur, indicating that the maturation step is located further downstream in the secretory pathway. For Δ-XylT the apparently unprocessed precursor (Figure 3C) co-localized exclusively with Golgi markers (Figure 3F), suggesting a post-Golgi compartment or, at the latest, the apoplastic space as the site for CLEL6 maturation. Maturation late in the secretory pathway was also observed for CLEL9. Similar to CLEL6, the Δ-KDEL and Δ-XylT constructs for CLEL9 were not processed and were retained in the ER and Golgi, respectively (Figure 3—figure supplement 1). For the Δ-Sec construct that is allowed to proceed beyond the Golgi, on the other hand, the smaller, processed product was generated (Figure 3—figure supplement 1). Our data thus indicate that both CLEL6 and CLEL9 mature after exit from the Golgi, in the TGN, in secretory vesicles, or in the apoplastic space. All CLEL peptide precursors including both CLEL6 and 9 share a conserved aspartate upstream of the sulfated tyrosine (Figure 1—figure supplement 1A). To test whether this aspartate is necessary for peptide processing and/or activity, we generated site-directed D-to-A mutants of both CLEL6 (D71A) and CLEL9 (D66A) and compared processing to the corresponding wild-type versions (Figure 4A,B). As compared to the fully processed Δ-Sec constructs of CLEL6 and CLEL9, the larger unprocessed form was observed for the Δ-Sec D71A and Δ-Sec D66A mutants, indicating that the aspartate is indeed required for processing (Figure 4A,B). When fused to the XylT Golgi anchor, the processing-resistant D71A and D66A mutants exhibited the same apparent molecular weight as the wild-type Δ-XylT constructs (Figure 4A,B), thus confirming that the band produced from the wild-type Δ-XylT construct of CLEL6 corresponds to the full-length precursor, despite its faster migration as compared to the unprocessed Δ-KDEL band (Figure 3C). Figure 4 with 1 supplement see all Download asset Open asset N-terminal maturation and the formation of bioactive CLEL6 and 9 peptides are aspartate-dependent. (A, B) The relevance of the N-terminal aspartate for precursor processing was analyzed on anti-GFP immunoblots for the secreted (Δ-Sec) and Golgi-anchored (Δ-XylT) constructs by alanine substitution (D71A and D66A for the CLEL6 and CLEL9 precursors, respectively). Aspartate-dependent processing requires SBT activity (Figure 4—figure supplement 1). (C) Complementation of the gravitropic response of the tpst-1 mutant by CLEL peptides produced in planta. The gravitropic response of the tpst-1 mutant (gray bar) was restored to wild-type levels (white bar) by treatment with cell wall extracts from plants expressing the CLEL6 or CLEL9 precursors (hatched bars). Activity was much reduced for extracts from plants expressing the D71A and D66A precursor mutants. Seedlings were grown for five days in the dark on ½ MS medium with peptides added as indicated. Plates were rotated 90° and gravicurvature was assessed after two days as the angle of the hypocotyl with the horizontal. Data show the average of three independent experiments as the mean ± SE (n ≥ 103). Unless otherwise indicated * and *** indicate significant differences to the tpst-1 control (gray bar) at p<0.05 and p<0.001, respectively (two-tailed t test). Figure 4—source data 1 Source data for hypocotyl gravitropic responses shown in Figure 4. https://cdn.elifesciences.org/articles/55580/elife-55580-fig4-data1-v1.xlsx Download elife-55580-fig4-data1-v1.xlsx A bioassay was then used to assess whether the aspartate and aspartate-dependent processing are required for the formation of bioactive CLEL peptides in planta. The full-length Sec CLEL6 and CLEL9 constructs were transiently expressed in N. benthamiana and any peptides produced from these precursors were extracted in apoplastic washes. The activity of these peptides was tested in the tpst-1 mutant, which is devoid of endogenous sulfated peptides (Figure 4C). When tpst-1 seedlings were treated with cell wall extracts of plants expressing sfGFP fusions of wild-type CLEL6 or CLEL9 precursors (GFP fluorescence was determined as a measure of protein expression, and equal amounts of GFP were used), the gravitropic response was restored to wild-type levels indicating the formation of bioactive CLEL6 and CLEL9 peptides (Figure 4C). In contrast, there was no bioactivity in cell wall extracts of plants expressing the alanine-substituted CLEL6 precursor, and reduced bioactivity in extracts from plants expressing the D66A-CLEL9 precursor (Figure 4C). The data confirm the importance of the aspartate residue for peptide maturation. The protease(s) required in addition to SBT6.1 for the final maturation step and activation of CLEL6 and CLEL9 was hitherto unknown. To test a potential involvement of SBTs, as suggested by the impaired gravitropic response of the hypocotyl in seedlings expressing EPI1a or EPI10 under the control of either the CLEL6 or the CLEL9 promoter (Figure 1A,C; Figure 1—fig

Plants modulate the efficiency of root nitrogen (N) acquisition in response to shoot N demand. However, molecular components directly involved in this shoot-to-root communication remain to be identified. Here, we show that phloem-mobile CEPD-like 2 (CEPDL2) polypeptide is upregulated in the leaf vasculature in response to decreased shoot N status and, after translocation to the roots, promotes high-affinity uptake and root-to-shoot transport of nitrate. Loss of CEPDL2 leads to a reduction in shoot nitrate content and plant biomass. CEPDL2 contributes to N acquisition cooperatively with CEPD1 and CEPD2 which mediate root N status, and the complete loss of all three proteins severely impairs N homeostasis in plants. Reciprocal grafting analysis provides conclusive evidence that the shoot CEPDL2 / CEPD1/2 genotype defines the high-affinity nitrate uptake activity in root. Our results indicate that plants integrate shoot N status and root N status in leaves and systemically regulate the efficiency of root N acquisition.

In plants, there are several kinds of intercellular signal transduction systems, such as with phytohormones and with peptide hormones. Small post-translationally modified peptides, which act as cell-to-cell signaling molecules, constitute the largest group of peptide hormones with various functions in intercellular signal transductions. We explored peptide hormones by in silico gene screening coupled with biochemical peptide detection. We also identified specific direct ligand-binding receptors for these peptides. In general, receptor genes are highly redundant in Arabidopsis and thus it is often difficult to identify the specific receptors by using classical forward and reverse genetics. To overcome this problem, we identify peptide hormone receptors by detecting direct peptide hormone-receptor interaction using photoaffinity labeling with photoactivatable peptide derivatives and custom-made receptor library. This review summarizes how we discovered these peptide hormone-receptor pairs and how these pairs function in plant growth and development.