Mobile Signaling Peptides: Secret Molecular Messengers with a Mighty Role in Plant Life

Abstract

Adaptive mechanisms for unfavorable environments have evolved in plants for thousands of generations, primarily in the form of endogenous chemical signals and the coordination of physiological processes. Signaling peptides (SPs) are diverse molecular messengers in various stress responses which have been identified in different plant families. SPs are recognized by the membrane-localized receptors and co-receptors, leading to downstream signaling for various plant responses. Progress in in silico analysis, along with other factors, has increased our understanding of the signaling peptide-mediated regulatory mechanisms underlying the entire plant life cycle. SPs mediate both long-distance (root-to-shoot-to-root) and local cell–cell communication via vascular system to communicate and coordinate with plant organs at distant locations. During abiotic stress, SPs inside plant cells perceive stress signals and transfer information at short and long physiological ranges through the signal transduction pathway, causing stress-responsive gene expression. SPs interact with pathogens and mediate cell-to-cell communication via signaling pathways. There are intriguing relationships between phytohormones and the secondary signaling cascades which are mediated by SPs. During biotic or abiotic stress, different peptides trigger jasmonic acid, ethylene, and ABA signaling, involving several secondary messengers. These messengers mediate the stress response via shared signaling components of ROS, Ca²⁺, and MAPKs, and they modify the gene expression for different phytohormones. In this review, we highlight current knowledge on the role of signaling peptides in plant adaptation, growth, and development. We aim to analyze the SP-receptor interactions and the significance of crosstalk between a few sample SPs and phytohormones. Potential directions on how scientists can use this information for crop improvement are also suggested.

Introduction

Signaling peptides (SPs), also known as ‘peptide hormones,’ are emerging growth hormones. These are small biological molecules of proteomes present in plants (usually < 20 amino acids when mature and rarely > 120 amino acids as a full-length precursor). SPs occur in very low concentrations at the physiological level (Olsen et al. 2002; Albert 2013; Matsubayashi 2014). Biosynthesis of these peptides involves proteolytic cleavage and post-translational modification, including tyrosine sulfation PHYTOSULPHOKINE (PSK), PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY), INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), ROOT GROWTH FACTOR (RGF) or CLEL/GLV and CASPARIAN STRIP INTEGRITY FACTOR (CIF) sulfated) by TPST, proline hydroxylation (Hyp) (proline-hydroxylated-systemin, CLAVATA 3 (CLV3)/EMBRYO SURROUNDING REGION RELATED (CLE), C-TERMINALLY ENCODED PEPTIDE (CEP), ROOT GROWTH FACTOR (RGFs), and PLANT PEPTIDE CONTAINING SULFATED TYROSINE 1 (PSY1), and hydroxyproline glycosylation (O-arabinosylation) (CLV3 and CLE2), in the primary structure of peptides for the generation of mature and active peptides for the bioactivation and perception of ligands (peptides) by receptors (Fig. 1) (Pearce et al. 2001a; Pearce and Ryan 2003; Ito et al. 2006; Kondo et al. 2006; Amano et al. 2007; Rojo and Denecke 2008; Ohyama et al. 2008, 2009; Komori et al. 2009; Matsuzaki et al. 2010; Tavormina et al. 2015; Patel et al. 2018). The plant genome contains several secreted peptide genes that encode SPs. In the model plant Arabidopsis thaliana, > 1000 genes encoding SPs have been reported, but only a limited number of signaling peptides have been characterized at the structural and functional levels (Lease and Walker 2006; Ghorbani et al. 2015). The secreted peptides orchestrate peptide-mediated cellular communication or signaling and bind to the corresponding plasma membrane-bound receptors (Czyzewicz et al. 2013). SPs act as ligands in this process, binding to specific membrane-localized receptors to make a ligand–receptor duo, which changes the configuration of receptors and activates their catalytic domains, whereas the spatial expression patterns of SPs lead to some specific interactions and functions in plants (Czyzewicz et al. 2013).

Fig. 1

General types of peptides: a Small post-translationally modified peptides and b Cysteine-rich peptides

Receptors are the main switches initiating downstream short-range and long-distance signaling in the cell and modulating cellular activities (Czyzewicz et al. 2013). However, temporal receptor expression and limited peptides restrict the activity of peptide signaling (Breiden and Simon 2016). Various small signaling peptides are yet to be functionally characterized (Czyzewicz et al. 2013). Several biochemical, genetic, and bioinformatics-based methodologies can help identify and characterize SP receptors in plants.

SPs play an essential role in plant reproduction, growth, and development, including homeostasis maintenance, stomatal aperture control (Takahashi et al. 2018a; Yu et al. 2018; Brito et al. 2018; Zeng et al. 2022; Fedoreyeva 2023), development of seeds (Doll et al. 2020), shoots (Brand et al. 2000), roots (Fletcher 2020; Shao et al. 2020), and pollen (Ge et al. 2017; Li et al. 2020) and, thus, yield (Yu et al. 2019), through specific mobile cell-to-cell communication (Matsubayashi et al. 2001). SPs and receptor kinase-mediated cell-to-cell communication play key roles in the initial stages of plant growth (Escobar-Restrepo et al. 2007; Okuda et al. 2009; Kessler et al. 2010). Plant intercellular communication is based on the signaling of non-peptide plant hormones (auxin, gibberellin, ethylene, abscisic acid, cytokinin, and brassinolides), small peptides, and specific receptors (Matsubayashi 2011). Small SPs interact with mobile transcription factors and noncoding RNAs, playing a crucial role in plant cell-to-cell communication (Vanneste and Friml 2009; Busch and Benfey 2010; Van Norman et al. 2011).

SPs act as silent heroes in biotic and abiotic stress defense and tolerance under diverse climatic conditions (Lindsey et al. 2002; Murphy et al. 2012; Albert 2013; Matsubayashi 2014; Ghorbani et al. 2014). During abiotic stress, SPs inside plant cells perceive stress signals and transfer signal information at short and long physiological ranges through the signal transduction pathway, causing stress-responsive gene expression (Akpinar et al. 2012). For example, SPs can interact with pathogens (Goyal and Mattoo 2014) and mediate cell-to-cell communication via signaling pathways (Murphy et al. 2012; Araya et al. 2014a, b; Tavormina et al. 2015). Cys-rich peptides are antimicrobial peptides owing to their antibacterial, antiviral, and antifungal (e.g., PR12 or defensins) properties in plant–microbe interactions for disease resistance (Hu et al. 2018). Rapid alkalinization factors (RALFs) in Arabidopsis act as negative regulators of the plant immune response to bacterial infection (Mang et al. 2017). Plant defensins (PDFs)—antimicrobial and host defense peptides—enhance fungal (i.e., Fusarium spp., Botrytis cinerea, and Verticillium dahlia) and bacterial (i.e., Pectobacterium carotovorum) resistance in model plants (Carvalho Ade and Gomes 2011; Ahmed et al. 2012; Gaspar et al. 2014). While numerous studies on SPs have been conducted, only few reviews have integrated the available information. Here, we have highlighted the current knowledge on how plants use these molecular networks and cues to respond to major biotic and abiotic stresses.

Signaling Peptides: Classification, Structure, and Function

Most SPs have been characterized in angiospermic plants, while symbolic SPs have been identified in green algae and mosses. Several peptide classes show high interspecific and intraspecific sequence diversity, while others contain single peptides or are only present in distinct plant families (Wheeler and Irving 2012). The first signal peptide molecule was discovered by isolating the 18-amino-acid systemin peptide (TomSys) from Solanum lycopersicum (tomato); this SP is restricted to Solanaceae family of plants (Ryan and Pearce 1998). Systemin regulates signaling events in systemic response, induces proteinase inhibitor biosynthesis in tomato leaves (Pearce et al. 1991) and production of jasmonic acid (Farmer et al. 1992a, b), defends plants against both the abiotic (salinity and UltraViolet (UV)) (Holley et al. 2003; Dombrowski 2003), and biotic (herbivory) stresses and also modifies root growth in plants (Ryan and Pearce 2003; Narváez-Vásquez and Orozco-Cárdenas 2008). In general, SPs are categorized on their structural characteristics as cysteine-rich peptides (CRPs) and small post-translationally modified (PTM) peptides (Fig. 1) (Matsubayashi 2012; Olsson et al. 2019). CRPs have even numbers (2–16) of Cysteine/Cys residues, with each CRP class having a distinct number and linear organization of amino acids. Mature CRPs are generally 40-amino-acid long but sometimes have > 100 but < 160 amino-acid residues and larger than small PTM peptides possessing intermolecular disulfide bonds, which determine the three-dimensional structure of mature proteins (Pearce et al. 2001a, b). CRPs are cationic with a conserved N-terminal. The C-terminal domain is Cys-rich, generally requiring 4–16 Cys residues to establish disulfide bridges to keep mature peptides in an active conformation (Fig. 1) (Pearce et al. 2001a). Disulfide bridges selectively degrade variable regions of pre-pro-peptides. The active peptide then becomes available for receptor binding. These peptides have several uniting structures regardless of higher amino-acid sequence divergence between individual peptides (Pearce et al. 2001a). CRPs act as antimicrobial compounds during plant–microbe interactions (Van Der Weerden et al. 2013; Tavormina et al. 2015), leaf stomata patterns and density, symbiosis, germination, guidance and burst of pollen tubes, plant gamete activation and seed development. CRPs are predominantly copious during plant reproduction. Due to their role in various reproductive processes, CRPs occur in female and male gametophytes, unlike PTM peptides that are mainly present in vegetative tissues (Hara et al. 2007; Sugano et al. 2010; Maróti et al. 2015; Bircheneder and Dresselhaus 2016). CRPs do not undergo post-translational modifications. They are further characterized into two subgroups based on proteolytic processing. The first subgroup includes CRPs that undergo proteolytic cleavage. For example, STOMAGEN/EPIDERMAL PATTERNING FACTORS (EPF)-LIKE9 (EPFL9) of the epidermal patterning factor (EPF) peptide family. STOMAGEN is a positive regulator of leaf stomata density (Hara et al. 2007; Kondo et al. 2010; Sugano et al. 2010). Another example is RAPID ALKALINIZATION FACTOR 1 (RALF1), recognized by the FERONIA (FER) receptor (Haruta et al. 2014; Pearce et al. 2001b), which also shows proteolytic cleavage and plays a role in cell expansion. The second subgroup contains CRPs that lack proteolytic cleavage such as S-locus cysteine-rich protein/S- locus protein 11 (SCR/SP11) and LUREs (Schopfer et al. 1999; Takayama et al. 2001; Okuda et al. 2009).

Small PTM peptides are small mature peptides (~ 10–20 amino-acid residues) produced by proteolytic processing and encoded by multiple paralogous genes. The longer translated precursor/propeptides correspond to mature peptides encompassing ~ 70–120 amino acids with few or no Cysteine/Cys residues, share conjoint tripartite structure, and undergo one or more PTMs in the C-terminal region; for instance, tyrosine sulfation, hydroxyproline glycosylation, and proline hydroxylation (Fig. 1). Non-CRPs have a tripartite structure of signal peptide at the N-terminal region, conserved C-terminal motif yielding the mature peptide, and frequently have proline residues in different genera within a plant family and a variable segment connecting the two (N and C) terminal domains (Matsubayashi 2014). PTMs increase the binding capacity of the signaling peptide to the receptor through structural conformation of peptides or direct interaction with receptors (Matsubayashi 2011). Non-CRP peptides form a major group of signaling peptides involved in plant growth and development through the regulation of many intercellular communication processes (Matsubayashi 2011). Examples include TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) (Matsubayashi and Sakagami 1999), ADVENTITIOUS ROOT FORMATION FACTOR (Amano et al. 2007), PLANT PEPTIDE CONTAINING SULFATED TYROSINE 1 (PSY1) (Amano et al. 2007), CLAVATA3CLV3/ EMBRYO SURROUNDING REGION RELATED (CLE) (Katsir et al. 2011), C-TERMINALLY ENCODED PEPTIDE (CEP) (Akker et al. 2016), and ROOT GROWTH FACTOR (RGF)/GOLVEN (GLV)/CLE-like (CLEL) (Fernandez et al. 2015), PHYTOSULFOKINE (PSK), and INFLORESCENCE DEFICIENT IN ABSCISSION (IDA). Table 1 lists the functions of various plant-signaling peptides.

Table 1 Functions of signaling peptides discovered in various plants

Identification of SP Receptors and Analysis of SP-receptor Interactions for Plant Signaling

The identification and functional annotation of SP receptors are key to understanding the mechanisms of signal transduction systems in plants. In the last two decades, various biochemical, genetics, and bioinformatics-based methods have been used to identify and characterize SP receptors in plant genomes, accelerating the discovery of SP receptors corresponding to secreted peptide hormones. Chow and McCourt (2006) defined hormone receptors based on their potential to recognize the subtle structural diversity between small molecules in a specific manner. As per this standard definition, molecular interactions between receptors and hormones should be non-covalent and reversible, producing an activated complex that induces the primary hormone response. From the receptor side, functional annotation of peptide signaling pathways can overcome the genetic redundancy of ligand peptides that are found exclusively as multi-gene families in plant genomes. Receptors are classified into three major categories based on their biochemical properties: two-component receptor system (TCS), leucine-rich repeat (LRR)-based receptors, and ubiquitination-based hormone receptors. Systematic classification of receptors allows researchers to compare their structure and function within plant hormone systems and other kingdoms (Chow and McCourt 2006).

TCS are well-known receptors in prokaryotes, fungi, slime molds, and plants (Stock et al. 2000; Ahmad et al. 2020). Members of this system play a principal role in regulating and modulating a diverse range of growth and developmental processes, including biotic and abiotic stress (particularly drought, high salinity, and high or low temperature) responses. TCS regulators were initially reported in prokaryotes and represent up to 1% of the genome size (Mizuno 1997). TCS systems of fungi and plants are more complicated than the canonical prokaryotic system (Chow and McCourt 2006). In this system, histidine kinases (HKs), an ancient and ancestrally conserved signaling mechanism, sense a signal input and response regulator that play an important role in output. In plants, TCS regulators typically comprise three types of proteins, including HKs, phosphotransfers (HPs), and response regulator proteins (RRs) (Ahmad et al. 2020). The functional members of TCS have been identified and characterized in the model plant Arabidopsis using different genomics methods, with three transmembrane HKs (AHK2, AHK3, and AHK4) characterized as cytokinin receptors (Schaller et al. 2008; He et al. 2020). These HKs respond negatively to biotic and abiotic stresses in Arabidopsis cytokinin signaling (Urao et al. 2000). In the transmitter functional domain, these three HKs perceive stimuli and are autophosphorylated at a conserved histidine residue, followed by the transfer of phosphoryl groups to HPs at a conserved residue of aspartate (He et al. 2016). Histidine–aspartate signaling mediated by TCS regulators controls a wide range of biological processes, including cell division, vascular differentiation, leaf senescence, male and female gametogenesis, seed development, and responses to environmental stimuli (He et al. 2016; Liu et al. 2020; Huo et al. 2020). TCS regulators are reported from several plants, including Arabidopsis, rice, maize, soybean, wheat, sesame, Chinese cabbage, tomato, cucumber, and Brassica rapa, among others (Pareek et al. 2006; Mochida et al. 2010; Chu et al. 2011; Liu et al. 2014; Gahlaut et al. 2014; He et al. 2016). However, studies are needed to determine the exact role and signal transduction mechanisms of each TCS element in plants. TCS signaling machinery in Arabidopsis is a complex system comprising 11 AtHKs, five AtHPTs, and 23 AtRRs (Hwang et al. 2002). Most TCS members in Arabidopsis interact with abscisic acid (ABA) in response to different abiotic stresses i.e., drought, salt, and low temperature (Nishiyama et al. 2013; Nguyen et al. 2016; He et al. 2016). The expression levels of three TCS elements (AHP1, AHP2, and AHP3) decrease significantly under heat stress (Miyata et al. 1998; He et al. 2020). Functional characterization of AHK1, a functional member of the AHK family, demonstrated its crucial role in stress responses, including drought, high salinity, and osmotic stress (Huo et al. 2020). Zhao et al. (2020a, b) reviewed the crosstalk between ethylene and other plant hormones focusing on rice and Arabidopsis mechanisms. Sharan et al. (2017) assembled an interactome map of TCS proteins in rice using a highly stringent Y2H platform and extensive in planta BiFC assays. The results revealed different sensory OsHK-mediated crosstalk in signaling. The same study reported OsHPTs as interaction hubs with OsRRs and highlighted interactions between type-A and type-B OsRRs, which led to a comprehensive molecular investigation of signal(s) transmitted by different TCS elements in a wide range of biological processes. Zhao et al. (2020a, b) analyzed a rice ethylene-response mutant mhz1 and reported that MHZ1 positively modulates root ethylene responses; MHZ1/OsHK1 works with the OsEIN2-mediated pathway to regulate root growth on ethylene perception. A more recent study demonstrated that HISTIDINE KINASE1 (HK1) coordinates the transfer cell specification in young barley endosperm (Hertig et al. 2020). Using RNA interference-mediated downregulation of Barley HISTIDINE KINASE1 (HvHK1), function of HvHK1 was investigated in ETC fate acquisition.

The leucine-rich repeat receptor kinase (LRR-RK) is one of the largest membrane integral receptor families evolutionary conserved in plants and animals. In plants, these receptors are essential for the intercellular signaling network as they perceive secreted hormone peptides (Chakraborty et al. 2019) and are involved in diverse external and internal signal responses to regulate plant growth and development (Ganie et al. 2021). Plant RKs are classified into 14 subfamilies based on ectodomain structure (Shiu and Bleecker 2001; Gou et al. 2010; Chakraborty et al. 2019). Previous genomic and biochemical studies report the XI class of the LRR-RK family as receptors of different SP signals, including CLE/TDIF (Ogawa et al. 2008; Hirakawa and Bowman 2015), IDA (Santiago et al. 2016), CEP (Tabata et al. 2014), Pep1 (Yamaguchi et al. 2006), RGF (Shinohara et al. 2016), PIP1 (Hou et al. 2014), and CASPARIAN STRIP INTEGRITY FACTORS (CIF) (Nakayama et al. 2017). While subclass X of the LRR-RK family is known for the PSK receptor PSKR (Matsubayashi et al. 2002). Through specific binding with ligands and hormones, LRR-RK controls diverse biological processes, such as cell division, proliferation, differentiation, and stem cell balance (Chakraborty et al. 2019). Recent efforts have identified novel LRR genes at the genome scale using a forward genetic approach. Three LRR-RKs (RGFR1, RGFR2, and RGFR3) were overexpressed in different root tissues, including the proximal meristem, elongation zone, and differentiation zone. In Arabidopsis, these three LRR-RKs directly interacted with the ROOT MERISTEM GROWTH FACTOR (RGF) peptide and regulated root meristem development (Shinohara et al. 2016). In Arabidopsis, cell wall LRR extensions (LRX) 3/4/5 are important for plant salt tolerance (Zhao et al. 2018a, b). Using immunoprecipitation–mass spectrometry (IP–MS), this study reported that LRX3/4/5 proteins interact with RALF peptides and defined an important signaling pathway for regulating plant growth and salt tolerance. Trihemasava et al. (2020) analyzed N-glycans on MIK1 and reported clusters of glycosylation that may explicate the regions participating in MIK1 ectodomain binding. Several studies have identified the crystal structure of different peptide-LRR-RK receptor pairs using X-ray crystallography and NMR (Song et al. 2016; Chakraborty et al. 2019). Peptide ligands act as molecular glue to stabilize the interaction between each corresponding receptor and its co-receptor (Fig. 2) (Morita et al. 2016). Song et al. (2016) reported the structure-guided discovery of RGF receptors for sustainable plant development. At a resolution of 2.6 Å, the same authors identified the crystal structure of RGF1 in a complex with LRR domain of RGFR1. Interaction between RGF1 and LRR domain suggests that the Arg-x-Gly-Gly (RxGG) motif is important for recognizing the sulfate group of RGF1 by RGFR1.

Fig. 2

Crystal structure of the probable LRR receptor-like serine/threonine-protein kinase At4g26540 (Chain B) with peptide PTR-SER-ASN-PRO-GLY-HIS-HIS-PRO-HYP-ARG-HIS-ASN (Chain A); a Hydrophobicity surface representation of the overall structure of hormone receptor RGFR1 in complex with RGF1 (PDB: 5HYX); b A close view of pocket with peptide structure in ball-and-stick model colored by atom types, yellow dashed lines indicate the hydrogen bond networks; c Peptide molecule as ball-and-stick model with interacting receptor residues labeled. This structure was rendered in different CPK using UCSF-Chimera, PyMol, and Ligplot + programs

Some plant hormones have unique signaling mechanisms, evidenced after discovering a cluster of genes responsive to exogenous auxin (Chow and McCourt 2006). In general, these types of genes encode two classes of molecules, Aux/IAAs and SCF (Skp1/Cullin/F-box) ubiquitin proteins (Rouse et al. 1998; Tian and Reed 1999; Nagpal et al. 2000; Ruegger et al. 1998; Gray et al. 2001; Rogg et al. 2001). Aux/IAAs belong to primary/early auxin-response genes, such as GH3 (Gretchen Hagen 3) and SAUR (small auxin up RNA) (Abel and Theologis 1996), which play a crucial role in the early stages of auxin signal transduction and respond quickly to auxin treatment. Functional members of Aux/IAAs mediate phytohormone signaling pathways in plants, including the jasmonic acid, salicylic acid, ethylene, and brassinosteroid pathways (Kazan and Manners 2009; Waseem et al. 2018).

The molecular weight of Aux/IAA proteins ranges from 18 to 36KD and is localized in nuclear compartment (Guifoyle and Hagen 2007). The number of Aux/IAA domains leads to classification of the whole family into four groups (I to IV). A typical domain of these proteins comprises 7–40 amino-acid residues (Gray et al. 2001), whereas on the basis of presence of four functional domains, Aux/IAAs are classified into canonical Aux/IAAs (with domains) and non-canonical Aux/IAAs (without domains). In general, Domain I of Aux/IAAs comprises LRRs (LxLxLx) that function as transcriptional repressors (Song et al. 2009). Domain II interacts with F-box proteins and plays an important role in Aux/IAA stability (Tiwari et al. 2004). Among Aux/IAAs and ARFs, Domains III and IV are responsible for homodimerization and heterodimerization (Rinaldi et al. 2012). The availability of whole-genome sequencing data in public repositories has accelerated genome-wide identification and functional annotation of Aux/IAAs. The auxin-insensitive mutant iaa1/axr5 revealed auxin-mediated growth effects in Arabidopsis (Yang et al. 2004). OsIAA9 or OsIAA13, a functional member of the Aux/IAA family, affects starch accumulation and participates in lateral root intonation in rice (Luo et al. 2015). Recently, at the TIR1 N- and C-termini, essential residues were identified with attractive interfaces for no-native interaction with IDRs followed by folded PB1 domain found in Aux/IAA (Niemeyer et al. 2020). Sun et al. (2020) produced a comprehensive protein–protein interaction map that mediates environmental and hormonal responses and cloned 113 phytohormone-related genes and 29 light signaling components in Arabidopsis. After individual testing, 141 interactions were identified using an Y2H approach. The results of these studies demonstrate that peptide–receptor compatibility is crucial for understanding peptide-mediated signaling in plants.

Post-translational Modifications of Signaling Peptides for the Orchestration of Peptide-Signaling and other Signaling Pathways

Signaling peptides are emerging growth hormones that mediate cell-to-cell signal transmission and regulate plant growth and development. The SPs act as ligands and make a ligand–receptor duo with the membrane-localized receptors, which are main switches that initiate the downstream signaling in the cell. Peptide biosynthesis often involves two major events: proteolytic cleavage and PTM. Analysis of peptide primary structure indicates that these two modifications are important to produce a mature and active peptide (Rojo and Denecke 2008).

Proteolytic Cleavage

The proteolytic cleavage is an important event in the life of precursor proteins to release an active and mature peptide. It is carried out with the involvement of different protease enzymes. The precursor proteins consist of a signal located at the N-terminus, which is cleaved by the peptidases once the precursor or pre-pro protein enters the endoplasmic reticulum (Tavormina et al. 2015). Proteolytic cleavage of the signal from N-terminus is necessary for further PTM. A peptide may be cleaved at single point or multi-points depending upon the location of peptide on pre-pro-protein. For example, in some of the RGF and CLE family peptides, situated at the C-terminus, proteolytic cleavage at only one point is sufficient to release the mature peptide (Fig. 3). In most cases, the peptide is located near the C-terminus and not at the exact end of the pre-pro-protein, and in such cases, proteolytic lysis at two points is needed to get a mature peptide (Ghorbani et al. 2015).

Fig. 3

A signaling peptide (SP). Nucleus-localized genes encode pro-preproteins peptides, undergoing cleavage processing for cleaving signal peptide in endoplasmic reticulum (ER). Maturation of peptides includes cleavage of the signal peptide (SP) in the ER during preproprotein synthesis, tyrosine sulfation by TPST in the Golgi apparatus (Komori et al. 2009), proline hydroxylation by prolyl-4-hydroxylase (P4H) and tri-arabinosylation by hydroxyproline O-arabinosyltransferase (HPAT) in the case of PSYs in the Golgi apparatus, and cleavage of the N- and C-terminals by subtilases (SBT) in the apoplast to release the mature peptide. Peptides are perceived at the plasma membrane by a receptor/co-receptor pair that mutually transphosphorylate each other and activate the receptor. LRR-RLKs-HEA and proton-pumping H⁺-ATPase (AHA) have been identified as direct targets of the peptide’s receptors PSKR1 and PSY1R. Modified peptides are predicted to regulate Pep-responsive genes (SPRGs) via unknown signaling intermediates and transcription factors (MYB15/MY36-CIFs), and via MAPK, Transphoprylation signaling pathway produces a biological response

Post-Translational Modifications (PTMs)

Till now most of the peptides reported are derived from non-functional longer precusor; and these peptides are grouped into three categories viz., (1) peptides subjected to specific PTMs, (2) Cys-rich peptides without PMT, and (3) non-Cys-rich peptides without PMTs (Tavormina et al. 2015). However, the post-translational modified peptides are derived from 70 to 120 amino-acid long pre-proproteins. Three types of PTM have been identified: tyrosine sulfation, proline hydroxylation (Hyp), and hydroxyproline glycosylation (O-arabinosylation) (Tavormina et al. 2015) (Fig. 1). While the enzymes responsible for PTMs have been identified, how they affect peptide bioactivity and signaling is unknown.

The first type of PTM is tyrosine sulfation, which occurs via peptide sulfation of tyrosine residues in most eukaryotes, including higher plant peptides. Peptides undergo sulfation in secretory pathways by a transmembrane enzyme, tyrosylprotein sulfotransferase (TPST), which transfers the sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the tyrosine residue (Moore 2003). Four peptide classes—PSKs (PHYTOSULFOKINES), PSYs (PLANT PEPTIDES CONTAINING SULFATED TYROSINE), RGF (ROOT MERISTEM GROWTH FACTORS), and CLEL (CLAVATA3/EMBRYO SURROUNDING REGION-LIKE) /GLV (GOLVEN), and CASPARIAN STRIP INTEGRITY FACTOR (CIF) are reportedly sulfated by TPST in tyrosine residues. The PSK and PSY precursor proteins are sulfated using TPST as the catalyzing enzyme and PAPS as the sulfur donor (Komori et al. 2009). Sulfation of tyrosine increases the hydrophilic binding of peptides, enhancing their affinity to bind more strongly to their receptors (Wang et al. 2015). For example, the binding of PSK to PSKR1 is mediated by H-bond formation and interaction of the sulfate group with tyrosine residue at the sub-domain of PSKR1 (Stührwohldt et al. 2011; Igarashi et al. 2012). Similarly, the binding affinity of unsulfated RGF peptides to RGFR1 decreased by approximately 200 times (Shinohara et al. 2016). These studies suggest the importance of sulfation for activating signaling peptides and peptide–receptor interactions. Because TPST plays a crucial role in tyrosine sulfation of signaling peptides, analysis of loss-of-function tpst mutants is useful for elucidating the bioactivity and impact of sulfated peptides on plant growth. Arabidopsis Attpst knockout mutants showed pleotropic phenotypes with dwarf roots, small hypocotyls, smaller veins, pale green leaves, premature senescence, and fewer flowers and fruits (Stührwohldt et al. 2011; 2015). However, all defective phenotypes could be recovered partially by applying sulfated peptides to tpst knockout plants (Komori et al. 2009; Doblas et al. 2017). Besides, plants with unsulfated and non-hydroxylated CLE8 peptides have altered lateral root growth and development; this phenotype was rescued by applying sulfated CLE8 (Matsuzaki et al. 2010). These results indicate the crucial role of tyrosine sulfation in signaling peptides for plant growth and development and stress resistance.

The second type of PTM is proline hydroxylation (Hyp), the most common and stable type in plant peptides, growth factors, and hormones (Myllyharju 2003). Various Hyp peptides, including HypSys (proline-hydroxylated-systemin), CLV3, CLE, CEPs, RGFs, and PSY1 have been isolated from plant tissues (Pearce et al. 2001a; Pearce and Ryan 2003; Ito et al. 2006; Kondo et al. 2006; Amano et al. 2007; Ohyama et al. 2008, 2009; Matsuzaki et al. 2010; Whitford et al. 2012; Patel et al. 2018). Accumulating evidence reveals that Hyp is crucial for peptide bioactivation and ligand perception (Mohd-Radzman et al. 2015; Patel et al. 2018). Peptides have been isolated in PTM (hydroxylated or glyoxylated) or unmodified (Patel et al. 2018) forms. This variation in peptide structure marks the significance of post-translational modification in the biogenesis of structurally different peptides or the inefficiency of PTM to make the peptides functionally active. Although Hyp is the most commonly occurring PTM in plants, the enzyme hydroxylating proline residues is unknown. Putative prolyl 4-hydroxylases (P4Hs) are involved in the biosynthesis of hydroxyproline-rich glycoproteins, including extensins, arabinogalactan proteins, and proline-rich proteins (Velasquez et al. 2015). Arabidopsis has 13 P4H-encoding genes, including those encoding enzymes for modifying precursor proteins (Myllyharju 2003; Matsubayashi 2012). P4Hs are Golgi-localized proteins (Yuasa et al. 2005), hydroxylating proline at different positions in precursor peptides, an initial step for further O-linked glycosylation (Pearce et al. 2001a; Amano et al. 2007; Ohyama et al. 2009). Among the Golgi-localized P4Hs, those which require P4H for peptide biogenesis have not been discovered. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a 14-mer peptide post-translationally hydroxylated at the ninth proline residue and functions in the abscission of senesced or damaged floral parts (Butenko et al. 2003). IDA acts as a ligand, inducing the expression of genes encoding LRR-RLKs-HAE and HAESA-like 2 (HSL2) receptors in the abscission zone (Santiago et al. 2016). Post-translational proline hydroxylation is important for the functional activation of IDA, its perception by receptors, and activation of the downstream-signaling cascade required for cell separation in the abscission zone (Schardon et al. 2016). Besides HAE and HSL2 receptors, IDA appoints SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERKs) which act as act co-receptors downstream of IDA. The ligand–receptor complex produced by IDA-HAESA-SERK generates a signal initiating a downstream-signaling pathway involving MITOGEN-ACTIVATED PROTEIN KINASE (MAPK). In turn, MAPK and MKK4/MKK5 act in the signaling pathway to regulate cell separation that leads to the abscission of floral parts (Stenvik et al. 2006; Cho et al. 2008; Meng et al. 2016). Analysis of the structural activity of the IDA and receptors show that proline hydroxylation at position 9 enhances IDA activity in Nicotiana bethemiana (Santiago et al. 2016). On the other hand, the abscission activity in floral parts remained unaffected in hydroxylated or non-hydroxylated IDA (Stührwohldt et al. 2018). Post-translational hydroxylation of proline residues at positions 4 and 11 is highly effective in the bioactivation of CEPs to increase legume nodulation (Imin et al. 2013; Mohd-Radzman et al. 2015). In contrast, modifying proline at position 11 with the tri-arabinosyl group decreases CEP activity (Patel et al. 2018), suggesting that not all PTMs are involved in peptide activation.

The last major type of PTM is hydroxyproline arabinosylation, common in several classes of abundant cell wall proteins and frequently observed in plant peptide hormones and growth factors (Kieliszewski et al. 2011). The enzyme involved in Hyp-arabinosylation is hydroxyproline O-arabinosyltransferase (HPAT), a golgi-targeted type II transmembrane protein which triggers the arabinosylation signaling cascade resulting in tri-arabinosylation at the hydroxyl group of proline. In Arabidopsis, three genes encode HPAT and hpat knockout mutants with loss-of-function of signaling peptides, and other cell proteins as well showed various defective phenotypes with cell wall thickness, altered flowering time, early senescence, hypocotyl elongation, and impaired pollen tube development (Ogawa-Ohnishi et al. 2013). CLV3 is 13-amino-acid long peptide, hydroxylated at seventh proline residue and triarabinosylated and is the first identified hydroxyproline-arabinosylated peptide growth factor purified from Arabidopsis plants overexpressing CLV3 precursor peptide. Similarly, another proline hydroxylated and triarabinosylated glycopeptide, CLE2, was purified from apoplastic extracellular fluids. Constitutive expression of CLV3 and CLV2 in knockout clv3 mutants revealed the importance of O-arabinosylation in the activation and maturation of CLV3/CLV2 peptides (Ohyama et al. 2009). CLAVATA3/Endosperm Surrounding Region (CLE) peptides are 13-amino-acid long peptides modified with triarabinose at Hyp at position 7. They are involved in the orchestration of signaling between roots and shoots to induce nodulation (Imin et al. 2018). The importance of glycosylation in CLE40 activity (Okamoto et al. 2013) and perception by ligands in soyabean was reported (Corcilius et al. 2017). Functional analysis of clv3 knockout mutants showed that the resumption of normal phenotypes occurs by complementation with glycosylated CLV3/CLV2 only, indicating that CLV peptides involve hydroxylation of proline residues and arabinosylation of hydroxyproline (Ohayama et al. 2009). Molecular studies suggest that triarabinosylation induces conformational changes in CLV3, influencing CLV3 binding specificity for receptors which was evaluated in an in vitro study of chemically synthesized CLV3, which interacts more strongly with CLV1 (CLAVATA1, receptor kinase) than non-arabinosylated CLV3 (Ohayama et al. 2009), restoring large meristems in the loss-of-function clv3 mutants upon exogenous application (Prusinkiewicz et al. 2007). These findings suggest that a considerable proportion of structurally related CLV peptides may be arabinosylated. Post-translational glycosylation affects peptide conformation through steric interactions with the peptide backbone, modulating the binding ability and specificity of peptides to target proteins (Seitz 2000). Tyrosine-sulfated peptides, including PSY1, CEPs, and HypSys, need additional modification of arabinosylation at Hyp residues to enhance their activity (Amano et al. 2007; Pearce and Ryan 2003). It is noteworthy that the first identified HypSys in tomato was found without Hyp or Hyp-glycosylation (Pearce et al. 1991, 2001a). These reports highlight that glycosylation plays a reversible (arabinosylation/dearabinosylation) role in regulating peptide activity and ligand-receptor binding.

Mobility of the Small Peptides: Short Range (intercellular) or Long-Distance (from source tissue to target tissue)

Signaling peptides mediate both long-distance (root-to-shoot-to-root) and local signal; cell–cell communication systems and peptide-based signaling communication are dominant in plants (Lease and Walker 2006; Oh et al. 2018). Signaling peptides can be mobile or bound to the membranes. Peptides are recognized by the receptors localized on membranes and co-receptors of shape-complementary nature. This ligand-receptor/co-receptor connotation starts signaling at intracellular levels for various plant responses (Kim et al. 2021). Signaling peptides regulate vascular development and abiotic stress responses by localized cell-to-cell communication pathways (Fukuda et al. 2007; Fukuda and Hardtke 2020; Kim et al. 2021). Tracheary Element Differentiation Inhibitory Factor (TDIF) produced in phloem translocate to cambium and attaches to the PHLOEM INTERCALATED WITH XYLEM/TDIF RECEPTOR (PXY/TDR) on the cell membrane (Hirakawa et al. 2008; Etchells et al. 2016; Fletcher 2020).

Long-distance signals transport rapidly through mass flow in xylem or phloem pathways. Thus, movement of peptides within vascular system allows one part to communicate and coordinate with plant organs at distant locations (Ham and Lucas 2017; Winter and Kragler 2018). Organ-to-organ communication mediated by long-distance signals in xylem and phloem is crucial to maintain homeostasis in plants (Caetano-Anollés and Gresshoff 1990; Oka-Kira and Kawaguchi 2006; Ruffel et al. 2011). There is a functional connection between xylem and phloem signaling pathways. Small mobile signaling peptides specifically are usually recognized and bind to an array of extracellular receptor domains of transmembrane proteins of receptor-like kinase family which are important components of their perception machineries. The peptide–receptor interaction triggers various processes at biochemical and physiological levels. SERK family receptor-like kinases act as co-receptors for the activation of peptide–receptor-like kinase pair through heterodimerization and transphosphorylation (Oh et al. 2018; Chen et al. 2020b).

Systemin signal peptides are also supposed to show long-distance signaling by transport through phloem (Chen and Kim 2006; Lough and Lucas 2006). Signal peptides applied exogenously to stems transport through xylem to the leaves (Pearce et al. 2001a, b; Scheer et al. 2005; Huffaker et al. 2006). The SHORTROOT protein is synthesized in the xylem, procambium, and pericycle of roots and transported locally to phloem poles and quiescent center to regulate development of phloem (Kim et al. 2020). Arabidopsis CLE genes encode peptides which are recognized by transmembrane receptors at the cell surface and in turn trigger signal transduction at intracellular level. This regulates growth and development in plants (Yamaguchi et al. 2016; Fletcher 2020; Fukuda and Hardtke 2020). The CLAVATA3/ESR-related 25 peptides (CLE-RS) and C-terminally Encoded Peptide (CEP) arrays are translocated from the root vascular system (xylem) as long-distance (root to shoot) mobile signals of drought or Nitrogen starvation to the shoot wherein ascending signals are recognized and directly bound to membrane-associated receptor kinases (HAR1, CLV1-homologous receptor) and CEP receptors, respectively (Okamoto et al. 2013; Tabata et al. 2014; Takahashi et al. 2018a, b, c). A CLE-RS receptor (HAR1) and CEP1 receptor (XIP1/CEPR1) are specifically expressed in the phloem (Nontachaiyapoom et al. 2007; Bryan et al. 2012). The peptides derived from roots are converted to secondary signal messengers in phloem and transported via phloem sap from “shoot-to-root” (Sasaki et al. 2014). The ascending signals trigger shoot–to-root secondary signals and induce systemic response of plant roots to upregulate nitrate transport. In nitrogen status signaling network, plant hormone cytokinin operates alongside CEP DOWNSTREAM peptides which send long-range communication signals from shoot to root through phloem (Ruffel et al. 2016; Ohkubo et al. 2017; Poitout et al. 2018). CLE-RS/HAR1 cascade plays vital role in autoregulation of nodulation in legumes (Sasaki et al. 2014).

The CEP genes upregulate in response to N-starvation and express in lateral root stele. Then CEP family peptides translocate from xylem vessels to shoots wherein these are recognized by the CEP Receptor 1 (CEPR1) kinase of leaf vascular tissue (Roberts et al. 2013; Delay et al. 2013; Tabata et al. 2014). The putative shoot-derived secondary signals upregulate nitrate transporter gene (NRT2.1) in plant roots to compensate for local N-starvation (Roberts et al. 2013; Delay et al. 2013). CEP-CEPR-signaling module works in all seed plants. According to Takahashi et al. (2018a, b, c), CLE25 is drought activated to mobile signal which moves from plant roots to the leaves wherein these are perceived by BAM1 and BAM3 receptors and CLE25 interactions activate carotenoid-cleaving enzyme NCED3 expression. NCED3 generates an ABA precursor molecule, active ABA signal, which enables plant to cope with a water shortage (Nambara & Marion-Poll 2005).

Signaling Peptide-Mediated Regulation of Plant Growth and Development

Small SPs help regulate plant growth and development through specific mobile cell-to-cell communication (Brand et al. 2000; Matsubayashi et al. 2001; Brito et al. 2018; Zeng et al. 2022; Fedoreyeva 2023). Knowledge of signaling peptide-mediated regulation pathways underlying the whole-plant life cycle is expanding, mostly due to progress in in silico analysis, in vitro design, and in planta verification (Murphy et al. 2012; De Coninck and De Smet 2016; Boschiero et al. 2020; Fletcher 2020). Here, we focus on biological function, interactions, and crosstalk between some representative SPs and phytohormones, target receptors, and downstream changes to illustrate their role in plant growth and development (Table 2).

Table 2 Signaling peptide-mediated regulation pathway and biological function in plant growth and development

Seed Development

Seed development requires deep crosstalk between the embryo and endosperm (Moussu et al. 2017). During seed development, the embryonic cuticle serves as a hydrophobic barrier for de novo deposition of the embryo, likely controlled by a signaling pathway involving ABNORMAL LEAF SHAPE1 subtilase (ALS1) and two GASSHO receptor-like kinases (GSO1 and GSO2) (Creff et al. 2019; Doll et al. 2020). A recent report showed that a sulfated peptide TWISTED SEED1 (TWS1) acts as a GASSHO ligand in the embryo, but ALS1 modulates its precursor in the neighboring endosperm to release active peptides (Doll et al. 2020). Thus, the signaling peptide TWS1 mediates a bidirectional molecular dialog between the embryo and endosperm before seed germination.

Shoot Development

The CLAVATA3 (CLV3) peptide is a well-known and major regulator in shoot apical meristem (SAM), determining stem cell fate in the division of aboveground leaves, stems, and floral parts (Brand et al. 2000). The secreted peptide CLV3 from the outermost meristem cell layers binds with the CLV1 receptor (containing tandem LRR domain) in deeper cell layers to repress the expression of homeodomain transcription factor WUSCHEL (WUS), restricting stem cell expansion (Yadav et al. 2011). In contrast, WUS promotes the expression of CLV3 and, thus, leads to a negative feedback signaling pathway in shoot growth and development by modulating CLV1 and WUS in flowering plants (Murphy et al. 2012). However, a recent paper reported that CLV3/CLE is a haploid stem cell-promoting signal in the liverwort Marchantia polymorpha, suggesting a critical role in the evolution of land plants (Hirakawa et al. 2020).

Root Development

In the root apical meristem (RAM), CLV3/EMBRYO SURROUNDING REGIONRELATED (CLE) peptides act synergistically with a set of specific receptors, hormones, or other catalytic substances (Fletcher 2020). Similar to the CLV3 signaling pathway, CLE40 interacts with the RLK ARABIDOPSIS CRINKLY4 (ACR4) to activate the expression of WUSCHEL-related homeobox 5 (WOX5) in controlling cell meristematic activity (Murphy et al. 2012). A recent study suggested that ginsenosides modulate a novel PgCLE45–PgWOX11 regulatory loop for adventitious root branching (Liu et al. 2020). Furthermore, RAM involves crosstalk between CLE members and cytokinin. CLE10 inhibits root protoxylem differentiation by repressing the expression of ARABIDOPSIS RESPONSE REGULATOR (ARR5 and ARR6) to negatively regulate cytokinin signaling (Cammarata et al. 2019). In addition, the peptide ROOT GROWTH FACTOR 1 (RGF1) binds with LRR-RLK INSENSITIVE (RGI) to modulate the expression of PLT1 and PLT2, thereby guiding the YDA-MKK4/MKK5-MPK3/MPK6 cascade for primary root growth (Song et al. 2016; Shao et al. 2020).

Pollen Development

During the meeting of sperm and ovule by pollen tube delivery, 5-kDa cysteine-rich peptide RALFs and their RLK1-like receptor kinases (RLK1Ls) modulate pollen growth via the autocrine and paracrine signaling pathway (Ge et al. 2017). Two secreted peptides (RALF4 and RALF19) are autocrine ligands of BUPS1/2–ANX1/2 receptor complexes that maintain pollen tube integrity; when sperm enters the pollen tube for fertilization, another peptide (RALF34) acts as a paracrine signal competing with RALF4/19 to induce timely tube rupture for the release of sperm (Ge et al. 2019). In addition, the grass-specific EXINE PATTERN DESIGNER 1 (EPAD1), containing N-terminal signaling peptide and C-terminal glycosylphosphatidylinositol (GPI)-anchor sites, can bind with plasma membrane lipids to form primexine for pollen exine (Li et al. 2020).

Yield

Yield formation mediated by signaling peptides requires a full-screen signaling crosstalk at whole-plant growth and development stages. Similarly, two secreted signaling peptides (EPFL2 and EPFL9) coordinate ovule patterning and seed number with gynoecium and fruit growth by binding with ERL1 and ERL2 (LRR receptor-like) kinases (Kawamoto et al. 2020). Correspondingly, heterologous expression of soybean phytosulfokine (GmPSK1), a sulfated pentapeptide hormone with the sequence YIYTQ, markedly increased seed growth (seed size and weight) and yield in transgenic Arabidopsis and tobacco (Yu et al. 2019). Understanding the signaling peptide-mediated regulation pathway in growth and development might help improve yield production.

Abscission

The phytosulfokine (PSK) regulates fruit abscission on the activation by subtilisin like proteinase (SBTs) known as phytaspase 2, and provides insight into the induced expression of hydrolases for the degradation of cell walls in the abscission zones of the flowers and fruits of tomato plants (Reichardt et al. 2020). Inflorescence Deficient in Abscission (IDA)-like peptides and HAESA (HAE) and HAESA-LIKE2 (HSL2) receptor-like kinases are reported to be associated with abscission of tomato flowers (Lu et al. 2023) floral organs and cauline leaves in Arabidopsis thaliana (Patharkar and Walker 2018; Shi et al. 2019), corolla abscission in flowers of Nicotiana benthamiana (Ventimilla et al. 2021) and IDA-LIKE genes, RbIDL1 and RbIDL4 regulate petal abscission in Rosa bourboniana (Singh et al. 2023). Both PSK and IDL6 induce tomato pedicel abscission (Li et al. 2021).

Role of Peptide Signaling in Biotic Stress

Diverse pathogenic microorganisms threaten plants (e.g., bacteria, fungi, viruses, and oomycetes), endangering their existence and efficiency (Cramer et al. 2011). These pathogens decrease annual crop production and pose a serious threat to food security. Plants use diverse defense mechanisms to defend against enemies to survive or retain their efficiency (Roux et al. 2014; Ganie and Reddy 2021). Pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are two forms of plant immunity. In general, pathogen-associated molecular patterns (PAMPs) comprise microbial or pathogenic assemblies such as flagellins, lipopolysaccharides, and fungal cell wall components (chitins and glucans), recognized by unique plant receptors called pattern recognition receptors (PRRs), which auxiliary activate PTII (Zipfel and Felix 2005).

Furthermore, microbial pathogens secrete effector proteins, recognized by resistance (R) proteins that accelerate the activation of induced resistance responses (ETI) (Dangl and Jones 2001). The effector proteins are essential components of the fungal pathogen’s virulence alongside plants and are significant during the biotrophic process of infection (Sonah et al. 2016). The importance of pathogenesis-related (PR) proteins during plant–fungal pathogen interactions has been documented, with an increasing list of known pathogen effector proteins that interact precisely with PR proteins through infection (Breen et al. 2017). Within plant species, the complexity and effectiveness of the plant protection mechanism for combating pathogen attacks vary (Jones and Dangl 2006; Segonzac and Monaghan 2019). Several PR proteins are classified as antimicrobial peptides (AMPs). They typically have a wide range of antimicrobial activity as cysteine-rich molecules, including the families of PR6 proteins (proteinase inhibitors), PR12 proteins (plant defensins), PR13 proteins (plant thionins), and PR 14 proteins (lipid transfer proteins). AMPs are ubiquitous and form a significant part of the host defense against many microbial pathogens and pests in various living forms, from microbes to plants (Egorov et al. 2005).

Antimicrobial Peptides and Disease Resistance

Antimicrobial peptides (AMPs) are abundant in eukaryotic organisms as various types of PR peptides (Bulet et al. 2004). Usually, their mode of action involves disrupting the pathogen membrane in particular and non-specific electrostatic and hydrophobic connections with cell surface groups (Thevissen et al. 2003). AMPs are receiving more attention for enhancing disease resistance due to their all-rounder efficiency against several biotic stresses, such as bacterial, viral, fungal, and their function in abiotic stress tolerance. For example, the transcript levels of AMPs in tomato plants increase after bacterial and fungal infections, suggesting their role in disease resistance (Chan et al. 2005). PR6 peptides have demonstrated efficient antimicrobial activity against various fungal pathogens in in vitro studies (Terras et al. 1993). The most important antifungal peptides in plants are the PR12 or plant defensins. In vitro experiments have shown that plant defenses demonstrate antifungal activity against many fungal pathogens (Terras et al. 1995; Jha and Chattoo 2009). Furthermore, overexpression of plant defense peptides has had improved and long-lasting disease tolerance in model and crop plants (Anuradha et al. 2008; Ghag et al. 2012; Kaur et al. 2016). PAMP is commonly used to refer to molecules that induce natural immune responses. PAMPs are evolutionarily conserved pathogen-derived molecules that differentiate hosts from pathogens, as classically defined. They include lipopolysaccharides, bacterial flagellins, peptidoglycans, and yeast mannans. However, since these molecules are often synthesized by nonpathogens, the term ‘pathogen-associated’ is a contradiction, and a more precise term would be ‘microbe-associated molecular pattern.’ Therefore, it makes sense that hosts will have identified receptors for truly pathogen-specific molecules, but only in plants if their conclusive evidence of immune receptors that recognize virulence-related pathogen-encoded molecules such as type III effectors. The expression ‘microbe-associated molecule(s)’ is used here instead of ‘PAMP.’

Arabidopsis reacts to flagellin and a highly conserved flagellin protein fragment of 22 amino acids called Flg222 (Felix et al. 1999). Flg22 activates a signal transduction cascade, containing a MAP kinase cascade, transmembrane LRR receptor kinase (FLS2), so-called WRKY transcription factors, and downstream effector proteins (Asai et al. 2002; Gomez-Gomez and Boller 2000, 2002) (Fig. 4). Studies have shown that fungal pathogenicity in plants imposed a conserved MAPK signaling cascade homologous to the filamentation/pheromone response pathways in Saccharomyces cerevisiae (Turra et al. 2014; Xu and Hamer 1996). The processes regulating MAPK signaling through the fungus–plant interaction are mostly unidentified (Turra et al. 2014). However, Masachis et al. (2016) found that F. oxysporum increases extracellular pH during plant infection, activating the pathogenicity-related MAPK signaling cascade, thus, furthering invasive hyphal growth and virulence.

Fig. 4

A general schematic of plant cell responses to biotic stress. Signaling pathways downstream of PRRs in plants. Plants have receptor family like kinases, such as the FLS2 flagellin receptor. Although the overall structure of the FLS2 signaling pathway appears similar to the animal PRR signaling pathway, no individual components are preserved, and the similarity most likely reflects the ubiquity of eukaryotic MAPK stress-response cassettes that respond to environmental signals. The graph has been modified from Rejeb et al. (2014)

RALF peptides include four conserved Cys residues that can verify two disulfide bridges; they were first identified in tobacco (Nicotiana tabacum) for their ability to trigger rapid extracellular alkalinization in suspension-cultured cells (Pearce et al. 2001b). RALF peptides were subsequently found ubiquitous in the plant kingdom, with 37 members recognized in the Arabidopsis thaliana genome alone (Sharma et al. 2016; Campbell and Turner 2017). Plant RALFs are secreted as pre-propeptides, discharging the mature peptide by proteolytic processing (Srivastava et al. 2009). RALF peptides are involved in the signal sequence for extracellular extrusion and also contain special amino-acid motifs, such as the RRILA motif for S1P protease recognition (Srivastava et al. 2009) and YISY motif, important for signaling cascade activation (Pearce et al. 2001b) (Fig. 5). Furthermore, four conserved cysteines form two disulfide bonds that stabilize mature RALF proteins. Therefore, RALF peptides are classified into four major groups (Campbell and Turner 2017): Groups I, II, and III encompass typical RALF peptides, while Group IV contains the most deviating RALF peptides, missing RRILA and YISY motifs, and, in some cases, only three cysteines. RALF peptides engaged in plant growth and development regulation, including root cell elongation, pollen tube growth, and stress responses (Ge et al. 2019; Haruta et al. 2014). In recent years, studies have shown that plant–microbe interaction control involves RALF-mediated signaling. Stegmann et al. (2017) stated that RALF peptides in Arabidopsis act as negative regulators of the plant immune response to bacterial infection, as the binding to the FER receptor of processed RALF23 prevents the formation of the complex between kinases of the immune receptor FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) with their co-receptor BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED KINASE 1 (BAK1), essential for initiating immune signaling. Furthermore, ANXUR1 (ANX1) RALF receptors, the nearest FER homolog, are associated with PRRs and nucleotide-binding domain leucine-rich repeat (NLR) type R proteins to inhibit PTI and ETI (Mang et al. 2017).

Fig. 5

Schematic representation of a comparative account of the signaling mechanisms mediated by signaling peptides and phytohormones in plant cells. Peptide and hormone-mediated signaling (A-B) resemble their receptors, which are mostly leucine-rich repeats (LRRs) with receptor-like kinases (RLKs; co-receptors) that transduce secondary signals in the cytoplasm. The secondary messengers and other signaling components commonly include ROS, Ca²⁺ influx, and MAPK activity associated with both pathways (A–B)

PLANT DEFENSINS (PDFs), with about 5 kDa small peptides, appear to be the best-studied cysteine-rich peptides and AMPs (Tavormina et al. 2015). PDFs are distributed extensively and one of the largest and most complex protein families associated with pathogenesis in monocots and dicots (van Loon et al. 2006). PDFs are primarily antifungal, while some have reported antibacterial activity. The heterologous overexpression of different PDFs improved resistance to various fungi and bacteria in both the model plants and crops (Carvalho Ade and Gomes 2011; Gaspar et al. 2014). PDFs are internalized or connected to intracellular targets by the fungal cell or remain outside the cell and stimulate cell death by triggering a signaling cascade (Vriens et al. 2014). As such, they represent a typical example of host defense peptides acting more specifically than the classically suggested specific lipid bilayer disturbance for AMPs (Wilmes et al. 2011).

The Arabidopsis genome has ~ 285 PDF and defensin-like genes (Mondragon-Palominoet al. 2017). PDFs have been involved in various model plants and crops, displaying their participation in inherent immune responses to fungal pathogens such as Fusarium spp., Botrytis cinerea, and Verticillium dahlia, and bacterial pathogens, such as Pectobacterium carotovorum (Gaspar et al. 2014; Ahmed et al. 2012). PDFs have shown antimicrobial activity because they bind host intracellular targets that activate defense signals, such as inducing cell death, and interact with different fungal sphingolipids and phospholipids (Tavormina et al. 2015). Table 3 and Fig. 6 summarize peptides associated with biotic stress responses.

Table 3 Summary of peptides associated with plant defense response

Fig. 6

Role of some signaling peptides in biotic stress

Role of Peptide Signaling in Abiotic Stress

Peptide hormones are involved in plant adaptation to abiotic stress (Fig. 7). They are encoded by small coding genes, secreted from cells, and translocated to other targeted plant cells, where they bind to a receptor protein (Matsubayashi and Sakagami 2006; Matsubayashi 2014; Tavormina et al. 2015) and induce physiological responses (Matsubayashi 2014; Tör et al. 2009). Several peptide hormones function as long-distance signal molecules in organ-to-organ communication (Okamoto et al. 2013, 2015). Characterization of hormone-like signaling peptides in abiotic stress responses (e.g., drought, heat, salinity etc.) is in its infancy.

Fig. 7

Role of some signaling peptides in abiotic stress

Salinity Stress

CAP-DERIVED PEPTIDE 1 (CAPE1) belonging to CRP family is involved in the salinity stress response in Arabidopsis thaliana (Chen et al. 2014; Chien et al. 2015). AtCAPE1 (11-amino-acid) induced germination, produced yellowish cotyledons, and decreased growth under high salinity in A. thaliana, and PROAtCAPE1 is downregulated under salinity conditions (Chien et al. 2015). AtCAPE1 peptide and CEP negatively regulate plant salt tolerance response under high salinity by suppressing salt tolerance genes namely, DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and GALACTINOLSYNTHASE2 (GolS2)) (involved in production of osmolytes), ALDEHYDE DEHYDROGENASE 7B4 (ALDH7B4) (for detoxification), ABSCISIC ACID–RESPONSIVE ELEMENT BINDING PROTEIN 1 (AREB1) and ABA INSENSITIVE 5 (ABI5)) (regulates stomatal closure), and RESPONSIVE TO DESICCATION 20/CALEOSIN 3 (RD20/CLO3) (protects the plasma membrane) (Tavormina et al. 2015; Chien et al. 2015). CAPE1 (proatcape1) knockout mutants in Solanum lycopersicum tolerated high salt stress whereas PROAtCAPE1 overexpression or exogenous application of AtCAPE1 peptide restores the salinity response in it (Chien et al. 2015).

The CEP peptide family shows differential regulation under salt stress (Delay et al. 2013; Aggarwal et al. 2020). CEP knockdown improves salinity stress tolerance partially in cep3 mutant of Arabidopsis thaliana which had longer primary roots (Ohyama et al. 2008), while CEP3 overexpression decreased primary root length and increased shoot length and salinity tolerance (Delay et al. 2013). Systemin, CEP, the C-terminus of a cysteine-rich secretory protein antigen 5, and genes within the pathogenesis-related 1 protein (CAP) superfamily are associated with the salinity stress response (Orsini et al. 2010; Delay et al. 2013; Chien et al. 2015). When overexpressed in plants, systemin positively regulates salinity stress tolerance, whereas C-TERMINALLY ENCODED peptide (CEP) and CAP GENE FAMILY DERIVED Peptide (CAPE) negatively regulate salinity stress tolerance GRIM REAPER PEPTIDE (GRIp) induced oxidative stress and reactive oxygen species (ROS)-dependent cell death in Arabidopsis thaliana (Wrzaczek et al. 2015). Several defense-responsive peptides modulate salinity tolerance. Systemin peptide overexpression confers better salinity tolerance in Solanum lycopersicum (Pearce et al. 1991).

Overexpression of OSIP108 (oxidative stress-induced peptide) in Arabidopsis thaliana enhances oxidative stress tolerance (Spincemaille et al. 2014a, b). Plant Elicitor Peptides (PEPs) are involved in starvation stress and biotic stress. PEPs crosstalk with salicylic acid (SA), jasmonic acid (JA), and ethylene during abiotic stress (Huffaker et al. 2006; Flury et al. 2013; Liu et al. 2013; Tintor et al. 2013). AtPROPEP3 recognized by the PEP RECEPTOR 1 (PEPR1) receptor, induced salinity stress tolerance and biotic stress tolerance in plants in response to exogenous application with 13-synthetic AtPROPEP3/AT13 peptide fragments (KPTPSSGKGGKHN) in a culture-based salinity stress assay by regulating salinity-induced genes and activating Na⁺ influx (Nakaminami et al. 2018). Multiple small coding genes are involved in salinity stress-related signal cascades. AtPROPEP3 knock down makes Arabidopsis plants hypersensitive to salinity whereas AtPROPEP3 overexpression or exogenous application of AtPEP3 peptide induces tolerance to salinity (Nakaminami et al. 2018).

Salt stress leads to downregulation of Rapid Alkalinization Factor (RALF)1 in plant root system. On exogenous use of active RALF1 peptide which is perceived by FERONIA (FER) receptor, salinity stress toxicity increases due to Na + accumulation by inhibition of activities of ARABIDOPSIS H + -ATPASE 2 (AHA2) and Na + /K + transporters (Yu and Assmann 2018). The fer mutants are RALF1 insensitive and hypersensitive to salinity due to loss of cell integrity (Feng et al. 2018; Yu and Assmann 2018).

Salt conditions activate RALF22/23 peptide accumulation and which in turn on interaction with LEUCINE-RICH REPEAT EXTENSINS (LRX) regulates FER-mediated integrity of cell wall, ABA signaling, and Reactive Oxygen Species (ROS), and leads to salinity tolerance. Ca2 + signaling cascade is also maintained (Zhao et al. 2018a, b, 2020a; Feng et al. 2018).

Drought Stress

Drought stress causes serious reductions in crop productivity and plants employ various ways in response to this stress (Ganie and Ahammed 2021). SPs have been demonstrated over years to play important roles in minimizing the drought-induced damages in plants. The CLAVATA3/EMBRYO SURROUNDING REGION RELATED 25 (CLE25) peptides move from roots to leaves as long-distance mobile signal and controls stomatal closure. In leaves, CLE25 (12-amino-acid peptide) is recognized and binds to plasma membrane-localized BARELY ANY MERISTEM (BAM) 1 and BAM3 receptor-like protein kinases under dehydration stress. Induction of CLE25 gene expression modulates ABA accumulation in vascular tissues and NINE‐CIS‐EPOXY-CAROTENOID DIOXYGENASE 3 (NCED3) in Arabidopsis leaves (Christmann and Grill 2018; McLachlan et al. 2018; Takahashi et al. 2018b; Yoshida and Fernie 2018). The root-derived CLE25 peptide functions as a long-distance signal and transmits water-deficiency signals under dehydration through vascular tissues to induce stomatal closure due to more ABA production for decreasing water loss in leaves by transpiration and enhances dehydration stress resistance. The CLE25–BAM module functions as a signaling molecule for long-distance signaling under dehydration (Christmann and Grill 2018; McLachlan et al. 2018; Takahashi et al. 2018b; Yoshida and Fernie 2018). CLE9 peptide expression in guard cells of plant leaves has also an important role in ABA signaling-dependent drought response and salinity (Zhang et al. 2019). The overexpression of CLE9 in transgenic plants leads to stomatal closure and stronger resistance to water deficiency (Zhang et al. 2019).

CEP5 has antagonistic crosstalk with auxin signaling. CEP5-dependent signaling stabilizes AUX/IAAs transcriptional repressors to control the auxin response under drought and osmotic stress (Smith et al. 2020). Novel peptide-dependent control mechanisms contribute to the fine tuning of auxin signaling with a role in osmotic and drought stress tolerance. CEP5 phenotypes include auxin-mediated control of root architecture (Roberts et al. 2016). Exogenous CEP5 peptide application in seedlings and CEP5-overexpressed plants shows drought and osmotic stress tolerance in CEPR receptor dependent or independent mode (Smith et al. 2020).

Drought stress-induced phytosulfokine (PSK) regulates fruit abscission on the activation by subtilisin like proteinase (SBTs) known as phytaspase 2, and provides insight into the induced expression of hydrolases to degrade cell walls in the flower and fruit abscission zones of tomato plants (Reichardt et al. 2020). The overexpression proPSK1 or SBT3.8 (SUBTILISIN) transgenic plant positively regulates drought stress resistance (Stührwohldt et al. 2021).

Overexpression of the EPIDERMAL PATTERNING FACTOR (EPF) signaling pathway modified leaf stomata density and size, decreased transpiration, increased growth and biomass, and improved tolerance to drought and high CO₂ in Arabidopsis thaliana (Doheny-Adams et al. 2012). Excess Fe induces CASPARIAN STRIP INTEGRITY FACTOR 1 (CIF1) and CIF2, expressed in the root stele, binding to GASSHO1 (GSO1)/SCHENGEN3 receptor kinases, which control water and Fe permeability in the vascular stele of roots (Nakayama et al. 2017).

Nitrogen (N), Phosphate (Pi), Iron (Fe), and Sulfur (S) Stress

C-TERMINALLY ENCODED PEPTIDE (CEP) family acts as root-derived nitrogen (N)-demand signal in N-deficient soil conditions and ascends to shoots. The perception of CEP signals by leaf expressed XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR (CEPR1 and CEPR 2) produces putative shoot-derived phloem-specific polypeptides CEPD proteins. Putative shoot-derived phloem-specific polypeptides upregulate nitrate transporter genes in the roots (nitrogen sensing). If part of the plant root system is N-starved while the rest grows in N-rich soil, then the N-starved roots under nitrate deficiency use CEP hormones produced by root-derived ascending N-demand signals to signal distant CEPR receptors in shoots development (Mohd-Radzman et al. 2015; Ohyama et al. 2008; Tabata et al. 2014; Delay et al. 2013; Ohkubo et al. 2017). CEP1 functions in root-to-shoot signaling and regulates root development in plants under nitrate starvation (Tabata et al. 2014). CEPR1 and 2 perceive CEP1 to increase nitrate uptake in Arabidopsis (Ohkubo et al. 2017). In turn, CEP1-CEPR signaling induces CEP DOWNSTREAM-LIKE 1 (CEPDL1) and LIKE 2 (CEPDL2) proteins in leaves. CEPDL1 and CEPDL2 act as a leaf-derived phloem-mobile descending signals to N-rich roots, and induce nitrate transporter NITRATE TRANSPORTER 2.1 (NRT2.1) gene expression in the roots. The NRT2.1 in Arabidopsis thaliana mediates root N uptake and transport. CEP1 plays a role in all three N-stress tolerance processes. CEP1 overexpression in legumes inhibits lateral root emergence and enhances nodule development (Mohd-Radzman et al. 2015; Ohyama et al. 2008; Tabata et al. 2014; Delay et al. 2013; Ohkubo et al. 2017). When roots cannot uptake sufficient N, CEPDL2 and NRT1.5 and NRT2.1 are upregulated to absorb and transport N (Ota et al. 2020).

In Arabidopsis thaliana and Medicago spp., signaling by CEP-CEPR module regulates root system architecture, lateral root gravitropic set-point, shoot auxin content, and auxin transport to roots (Chapman et al. 2020). CLE1/3/4/7 expression increases in plants roots during N-starvation. There is increase in transcription levels of CLE3 which is recognized by CLAVATA (CLV1) receptor located in phloem. CLE-CLV1 signaling optimizes nitrate-dependent lateral root development, elongation, and emergence during N availability. CLV1-mediated N-demanding signal represses CLE-CLV1 cascade during low nitrate condition forming a feedback loop (Araya et al. 2014a, b; Chapman et al. 2020). The clv1 receptor mutant produces lateral roots under severe N deficiency due to the overaccumulation of CLE3 and CLE2 peptides. CEP peptide overexpression induced by low N reduces lateral root number in Medicago truncatula (Chapman et al. 2020). In Arabidopsis, ROOT MERISTEM GROWTH FACTOR 1 (RGF1, a 13-amino-acid peptide) is perceived by RGF1 INSENSITIVE 1–5 (RGI 1–5) receptors, and this cascade controls root development under phosphate (Pi) deficiency (Matsuzaki et al. 2010; Cederholm and Benfey 2015; Ou et al. 2016). CLE14 controls differentiation of the Root Apical Meristem (RAM) in P signaling (Gutiérrez-Alanís et al. 2017). In P. starvation environments, CLE14-CLV2/PEPR2 signaling attenuate POLTERGEIST (POLL) and POLTERGEIST-LIKE 1 (PLL1) which leads to downstream exhaustion of root meristems (Gutiérrez-Alanís et al. 2017).

In S starvation scenarios, expression levels of CLE2/3 peptide are controlled and repressed. CLE2/3 is perceived by CLV1 receptors (CLE-CLV1 module) to reduce lateral root density (Czyzewicz et al. 2015; Dong et al. 2019) but diminishes in clv1 mutants.

In Arabidopsis, CASPARIAN STRIP INTEGRITY FACTORS (CIF1 and CIF2 are 21 amino-acid peptides) function as a signal increase iron (Fe) tolerance by controlling Casparian strip formation and lignification in roots under excess iron (Doblas et al. 2017; Nakayama et al. 2017). GASSHO1 (GSO1)/SCHENGEN3 (SGN3) and GSO2 act as the receptor for synthetic CIF1. CIF1 and 2 restore iron homeostasis in cif1cif2 mutants by lignification of Casparian strip after treatment with these peptides (Doblas et al. 2017; Nakayama et al. 2017).

Heat Stress

The expression of CLE45 in floral stigma is activated by heat stress and mediates pollen tube growth wherein CLE45 is recognized by two receptors namely STERILITY-REGULATING KINASE MEMBER1 (SKM1) and SKM2 under heat conditions (Endo et al. 2013). Exogenous application of synthetic CLE45 peptide increases growth of pollen grain tubes under heat scenarios whereas skm mutants are insensitive to this treatment (Endo et al. 2013).

Do SPs and Phytohormones exhibit similar Signaling Mechanisms Accompanying Defense and Stress Tolerance in Plants?

The number of signaling peptides exceeds the number of conventional phytohormones in plants (Olsson et al. 2018), indicative of their myriad functions. In addition to phytohormones, SPs take part in short and long-distance signaling associated with developmental changes and stress sensing in plants (Olsson et al. 2018). Furthermore, various tissue-specific proteases are involved in precursor (prepeptides) processing, regulating the functional redundancy of SPs in plant tissues precisely with different ontogenic phases (Chen et al. 2019). During evolution, increasing genome complexity and diverse functions are accompanied by gene duplication for SP gene families in various plant species (Olsson et al. 2018). SPs also exhibit various forms produced by gene duplication, but some exhibit functional redundancy (Olsson et al. 2018). For instance, the CLE gene cluster produces different peptides with similar amino-acid sequences. CLE42 and CLE41/CLE44 inhibit tracheary element differentiation but do not inhibit root growth (Breiden and Simon 2016).

Despite the current knowledge on the role of SPs and their mechanisms of action in different plant biological processes, their differences with phytohormones in accomplishing these processes need deciphering. Emerging evidence suggests similarities in the mechanisms of perception, communication, and expression of SPs and phytohormones in plants subjected to normal or challenging environments (Chen et al. 2019). Nevertheless, future investigations may reveal some differences. SPs and other phytohormones have co-evolved in various plant groups, where increased structural and functional complexity has led to diverse functions and signaling routes (Olsson et al. 2018). Like phytohormones, SPs can move between the apoplast and symplast of cells (Stahl and Simon 2013).

Interesting connections exist between the secondary signaling cascades mediated by SPs and phytohormones. Various peptides elicit JA, ET, and ABA signaling during biotic or abiotic stress, involving several secondary messengers (Chen et al. 2019), which mediate the stress response via shared signaling components of ROS, Ca²⁺, and MAPKs, and modulate the gene expression for various phytohormones (Kandoth et al. 2007; Chen et al. 2019). IAA, JA, SA, ET, and ABA are the major phytohormones involved in crosstalk with various stress-induced peptides in plants (Chen et al. 2019). One or more PTM events are likely to regulate peptide activity; thus, peptides have a unique regulation system compared with other phytohormones. Unlike SPs, hormone signaling is regulated primarily by PTMs in their receptors or transcription factors (Gou and Li 2020; Semeradova et al. 2020). Similar to stress-induced hormone signaling, SPs bind specific cellular receptors (LRRs) to transduce the downstream cascade of secondary signals via co-receptors (RLKs), transcription factors, and various secondary messengers (Olsson et al. 2018). Thus, no remarkable differences exist for the downstream-signaling cascades mediated by peptides and hormones. Certain stress-induced SPs are involved in phytohormone crosstalk, either upregulating or downregulating their expression patterns (Chen et al. 2019). Systemins bind with SYSTEMOIN RECEPTOR 1 (SYR 1), triggering ROS accumulation, ethylene biosynthesis, and protease inhibitor expression. Furthermore, systemins trigger Ca²⁺ influx and MAPK phosphorylation to activate JA biosynthesis. PEP1 peptides trigger downstream defense responses to wound stimuli, involving JA, ET, and SA pathways accompanied by a burst of H₂O₂ (Bartels et al. 2013). Similarly, phytosulfokines (PSKs) induce Ca²⁺ signaling and mediate auxin biosynthesis to initiate necrotrophic responses (Zhang et al. 2018). Moreover, SPs also exert precise effects on root growth and architecture (Oh et al. 2018). CLE25 and CAPE1 exert associative effects on drought and salinity tolerance by regulating ABA homeostasis (Chen et al. 2019). Thus, it is evident that SPs and phytohormones operate at the crossroads of defense and stress tolerance signaling pathways in plants. While SP- and phytohormone-mediated signaling share some components, further investigations are needed to decipher the interaction of SPs with various other molecular components involved in hormone signaling pathways.

Noncoding RNA-encoded SPs

Previously, regulatory SPs were considered to be derived from precursor proteins as their processed products, ORFs via direct translation, and sometimes untranslated regions of mRNAs (Ren et al. 2021). However, many studies have documented that SPs are also encoded by noncoding RNAs (ncRNAs), including primary microRNAs (miRNAs), long ncRNAs, and circular RNAs (Legnini et al. 2017). Among the various ncRNA-derived SPs, only the role of pri-miRNA-derived peptides in regulating plant biological processes has been documented (Sharma et al. 2020). The pri-miRNAs possess a short ORF in their 5’ upstream region, which encodes regulatory peptides known as miPEPs (Ren et al. 2021), found in plant species such as grapes, Arabidopsis, soybean, and Medicago (reviewed by Ren et al. 2021). Endogenous miPEPs have been detected using the Western blotting technique and found to accumulate in plant tissues (Sharma et al. 2020). miPEPs may act as internal signals in a feedback mechanism to regulate miRNA accumulation in plants (Ormancey et al. 2020). These miPEPs are believed to control/activate pri-miRNA transcription, inducing mature miRNA upregulation and miPEP accumulation. The role of miPEPs in regulating the transcription of genes encoding pri-miRNAs has been reported in some plant species. For example, cordycepin (RNA synthesis inhibitor) supplementation inhibits the miPEP165a-mediated accumulation of pri-miR165a in Arabidopsis, indicating a positive effect of miPEPs on miRNA expression (Lauressergues et al. 2015). Similarly, GUS gene expression is activated by the miR858a promotor in two reporter lines of Arabidopsis, which is fused with only the start code or entire ORF encoded miPEP858a (Sharma et al. 2020). Sharma et al. (2020) reported that synthetic miPEP858a supplementation enhanced GUS activity, suggesting activation of the promotor and, thus, enhanced transcription of miR858a by miPEP858a. miPEPs may directly or indirectly function as trans-acting factors, such as transcription factors (TFs), to enhance miRNA gene transcription (Sharma et al. 2020). The actual regulatory mechanisms of miPEP activity remain unclear.

An investigation of pri-miRNAs in plants such as Arabidopsis (Sharma et al. 2020), grapevine (Chen et al. 2020a, b), Dimocarpus longan (Zhang et al. 2020) has revealed that several putative pri-miRNAs possess functional sORFs that code for regulatory micropeptides. The role of some miPEPs in plant biological processes has been elucidated. For example, exogenous application and overexpression of miPEP165a (in Arabidopsis) and miPEP171b (in Medicago truncatula) significantly induce the expression of their corresponding miRNAs, impairing main root growth and lateral root formation (Lauressergues et al. 2015). Similarly, application of miPEP171d1 in grapes upregulates MIR171d expression, and enhances adventitious root development (Chen et al. 2020a). Supplementation of synthetic miPEP172c increases nodule number in soybean roots (Couzigou et al. 2016). Sharma et al. (2020) demonstrated that miPEP858a regulates the phenylpropanoid pathway and auxin signaling genes, thus, controlling flavonoid biosynthesis and plant development.

Despite this progress in determining the biological function of miPEPs, many important questions remain unanswered. For example, do miPEPs specifically encode pri-miRNAs, and if so, how does it occur? There are few reports on the specific activation of miRNAs (Lauressergues et al. 2015). Application of synthetic miPEP171d1 to tissue-cultured grape plantlets reduces the expression of some miRNAs (MIR171i, MIR171a, and MIR160c), but it is not clear whether the incubation period causes this reduction (Chen et al. 2020a). Hence, studies are needed to elucidate whether miPEPs negatively affect their corresponding miRNAs or other miRNAs.

Does Peptide-Signaling Involve Multiple Crosstalk Events with Plant Growth Regulators? Future Prospects

Secreted peptides and membrane-bound receptors orchestrate cellular communication. Peptides bind to receptors and change their conformation, initiating downstream communication (for short-range and long-distance signaling) and modulating cellular activities and processes. Connections exist between the secondary signaling cascades mediated by SPs and phytohormones.

Higher plants contain several SP genes. Most SP research has been conducted on Arabidopsis, with > 1000 genes encoding SPs reported to date. However, few SPs have been structurally and functionally characterized. Peptide biosynthesis involves two major events—proteolytic cleavage and PTM—crucial for producing mature and active peptides. Proteolytic cleavage (single or multiple) is essential for releasing mature peptides as in RGFs, CLE, CEPs, and PSK families. However, the identified number of proteases in plants is less; further research is needed to identify more proteases. PTMs, including tyrosine sulfation, proline hydroxylation, and hydroxyproline glycosylation, are crucial for cell peptide biogenesis, activation, and subsequent downstream signaling. Several studies have demonstrated that PTMs contribute to plant adaptation to various biotic and abiotic stresses. In addition, PTMs of SPs have been associated with phenotypic plasticity related to roots, leaves, fruit ripening, flowering time, and early senescence.

Enzymes regulate the biosynthesis of various conventional phytohormones. However, questions remain unanswered about tissue-specific production and secretion mechanisms of SPs in plant tissues. In general, all plant cells can produce and deliver peptides, as with other phytohormones. SPs also exhibit dose-dependent reactions in cells. However, little information is available on the conjugation mechanisms or stability of SPs in plants.

Several questions remain unanswered on the mechanism of peptides which can trigger the functioning of secondary messengers and gene actions pertaining to hormonal actions. Thus, it is imperative to decipher the spatio-temporal regulation of the production of several peptides being originated from the precursor proteins under normal and challenging environment. The extent or intensity of response mediated by peptides and their regulation by a positive feedback loop should undergo detailed investigations. Future investigations are required to unravel the molecular signaling routes of various peptides under stress conditions in plants. It would be interesting to perform detailed investigations on the fate of mature peptides associated with cell secretion, transport, and long-distance signaling under abiotic stress. In this context, works for some insights into peptide-receptor interactions regulating specific developmental aspects will be worth to be undertaken in future. Challenges exist in deciphering the role of peptides involved in canonical signaling pathways to that of phytohormones. Furthermore, it would be interesting to investigate the associative role of peptides and novel plant growth regulators, including gasotransmitters (NO, H₂S, and CO), melatonin, and strigolactone. Thus, a holistic approach needs to be undertaken to extend our current understand of mechanisms of peptide formation, receptor recognition, long-distance signaling, and crosstalk with other plant-signaling molecules. Integrating various ‘omics’ technologies with SPs research would enhance existing knowledge, which could be translated into applied research for plant breeding and genetics.