Sulfated peptides and their receptors: Key regulators of plant development and stress adaptation

Abstract

Four distinct types of sulfated peptides have been identified in Arabidopsis thaliana. These peptides play crucial roles in regulating plant development and stress adaptation. Recent studies have revealed that Xanthomonas and Meloidogyne can secrete plant-like sulfated peptides, exploiting the plant sulfated peptide signaling pathway to suppress plant immunity. Over the past three decades, receptors for these four types of sulfated peptides have been identified, all of which belong to the leucine-rich repeat receptor-like protein kinase subfamily. A number of regulatory proteins have been demonstrated to play important roles in their corresponding signal transduction pathways. In this review, we comprehensively summarize the discoveries of sulfated peptides and their receptors, mainly in Arabidopsis thaliana. We also discuss their known biological functions in plant development and stress adaptation. Finally, we put forward a number of questions for reference in future studies.

Sulfated peptides and their receptors play critical roles in regulating plant growth, development, and environmental fitness. This article provides a comprehensive review of these peptides and their receptors, including their discovery, regulatory components, and biological functions. It also puts forward a number of key questions for future discovery studies.

Introduction

Intercellular communication is essential for coordinating growth, development, and environmental fitness in multicellular organisms. Accumulated evidence indicates that secreted peptide hormones play key roles in plant intercellular communication. For instance, CLAVATA 3, a peptide expressed in the shoot apical meristem, controls shoot apical meristem development (Fletcher et al., 1999). PLANT ELICITOR PEPTIDEs (PEPs), a group of wound-induced peptides, can activate plant defense responses (Yamaguchi et al., 2006). Under drought stress, the root-derived CLAVATA 3/EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) peptide can be transported from the root to the shoot to regulate stomatal closure in the leaves (Takahashi et al., 2018). Over 1000 genes encoding putative secreted peptides have been identified in the Arabidopsis genome (Lease and Walker, 2006; Stuhrwohldt and Schaller, 2019). On the basis of their structures, secreted peptide hormones are primarily categorized into two classes, cysteine-rich peptides and post-translationally modified peptides. Cysteine-rich peptides typically consist of 50–169 amino acids with 4–16 cysteine residues. Post-translationally modified peptides are usually generated from their precursor proteins with post-translational modifications such as proline hydroxylation, hydroxyproline arabinosylation, and tyrosine sulfation (Matsubayashi, 2014; 2018). The tyrosine sulfation modification relies on the enzyme TYROSINE PROTEIN SULFOTRANSFERASE (TPST; Komori et al., 2009). Only one copy of TPST has been found in the Arabidopsis genome. Previous studies have indicated that TPST is involved in root meristem maintenance, phosphate deficiency responses, and Casparian strip formation. On the basis of its biological functions, TPST has also been designated as ACTIVE QUIESCENT CENTER 1 (AQC1), HYPERSENSITIVE TO PI STARVATION 70 (HSP70), and SCHENGEN 2 (SGN2; Doblas et al., 2017; Kang et al., 2014; Matsuzaki et al., 2010; Zhou et al., 2010). Four types of sulfated peptides have been identified in Arabidopsis: PHYTOSULFOKINES (PSKs), PLANT PEPTIDE-CONTAINING SULFATED TYROSINEs (PSYs), ROOT MERISTEM GROWTH FACTORs (RGFs), and CASPARIAN STRIP INTEGRITY FACTORs (CIFs; Kaufmann and Sauter, 2019).

Although the functions of sulfated peptides were reviewed previously (Kaufmann and Sauter, 2019), significant progress has subsequently been made in related fields, and the information needs to be updated. The purpose of this review is therefore to present the latest research findings and to summarize our knowledge about sulfated peptide family members and their biological functions (Supplemental Table 1). This review is structured on the basis of the chronological discoveries of the peptides and focuses mainly on sulfated peptide signaling, particularly receptor recognition mechanisms, and important biological processes regulated by these peptides. We also discuss current research challenges and provide insights into future research perspectives.

PSK

PSK was the first sulfated peptide discovered in plants. Early studies showed that the growth rate of plant cells in liquid culture was correlated with their initial density. This limitation could be overcome by the addition of media from older cultures, suggesting that some substance secreted from plant cells can stimulate cell division, an observation that led to the discovery of PSK (Matsubayashi and Sakagami, 1996). Seven putative PSK-encoding genes were identified in the Arabidopsis genome. Some of their coded proteins, PSK1, PSK2, PSK3, PSK4, and PSK5 carry the canonical PSK motif YIYTQ, whereas PSK6 contains a PSK-related sequence, YIYTH. An additional putative PSK gene, At2g22942, was predicted to encode a protein with two typical YIYTQ motifs (Yang et al., 2001; Lorbiecke and Sauter, 2002; Kaufmann and Sauter, 2019). The precursor protein undergoes hydrolysis and two sulfonation modifications at two tyrosine residues to yield a biologically active PSK peptide. The bioactivity of PSK can be significantly enhanced up to 1000-fold through sulfation compared with its non-sulfated form (Matsubayashi and Sakagami, 1996; Kutschmar et al., 2009). PSK precursor genes show diverse expression patterns in various tissues at different developmental stages. In roots, PSK1 is strongly expressed in the epidermis, whereas PSK2, PSK3, PSK4, and PSK5 are detected in the central cylinder. PSK4 and PSK5 are expressed mainly in the endodermal cell layer. Only PSK1 and PSK3 are expressed in the primary root tips (Kutschmar et al., 2009). In aerial plant parts, PSK2, PSK3, PSK4, and PSK5 are broadly expressed in leaves, and PSK2, PSK4, and PSK5 are expressed in reproductive organs (Matsubayashi et al., 2006; Stuhrwohldt et al., 2015). The expression of PSK3 and PSK5 can be induced by injury (Loivamaki et al., 2010).

In Arabidopsis, PSK is recognized by PSK RECEPTOR 1 (PSKR1) and PSKR2, two members of the leucine-rich repeat receptor-like kinase (LRR-RLK) type X subfamily. The pskr1 pskr2 double mutant has a short primary root and is insensitive to exogenous PSK treatment (Kutschmar et al., 2009; Kaufmann et al., 2021). Overexpression of PSK1 or PSKR1 produces a similar root elongation phenotype (Matsubayashi and Sakagami, 2000; Matsubayashi et al., 2002, 2006). An extracellular 15-amino-acid island domain of PSKR1 (from Glu503 to Lys517) is responsible for PSK binding in Daucus carota (Shinohara et al., 2007). In Arabidopsis, the Arg300 and Asn346 of PSKR1 can form hydrogen bonds with the free carboxyl group of the Gln5 of PSK, and the Phe506 of the receptor is tightly bound to the Gln5 and Tyr3 of PSK. Two sulfate groups of PSK form hydrogen bonds with the Lys508 and Asn424 residues of the receptor, and the van der Waals pack PSK by the Leu399, Trp448, and Lys508 residues of the receptor. Mutations at any of these amino acids on the receptor can abolish its function (Wang et al., 2015). Although BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs) act as co-receptors in the PSK signaling pathway, they are not directly involved in the binding of PSK, in contrast to their roles in the brassinosteroid signaling pathway (Li et al., 2002; Nam and Li, 2002; Wang et al., 2015). A number of RLK-mediated signaling pathways have been studied extensively. Typically, reversible phosphorylation and dephosphorylation are key events in RLK-related signal transduction. Early studies pinpointed the significance of specific phosphorylation residues, such as Thr890, Ser893, Thr894, Ser686, Ser696, Ser698, and Tyr888, for PSKR1 function (Hartmann et al., 2014; Muleya et al., 2014, 2016; Kaufmann et al., 2017).

Besides its kinase activity, PSKR1 was also found to possess guanylate cyclase and CALCIUM-BINDING PROTEIN (CaM) activities (Kwezi et al., 2011; Hartmann et al., 2014). A rapid increase in CYCLIC GUANOSINE MONOPHOSPHATE (cGMP) levels was observed when 100 nM PSK was added to an Arabidopsis protoplast culture (Kwezi et al., 2011). In higher plants, cGMP has broad effects on various biological processes such as development, defense responses, and hormonal regulation (Leng et al., 1999; Newton et al., 1999; Kaplan et al., 2007). Treatment with physiological levels of Ca²⁺ can also enhance the guanylate cyclase activity of PSKR1-promoted cGMP production. The resulting cGMP can then inhibit phosphorylation of PSKR1 (Muleya et al., 2014). Several sites have been shown to influence PSKR1 function, including the GTP recognition site Gly923, the essential guanylate cyclase activity site Gly924, the ATP-binding site Lys762, and the CaM-binding site Trp831. The function of PSKR1 requires synergistic kinase, guanylate cyclase, and CaM-binding activities. Substitutions of key residues related to these enzymatic activities result in loss of PSKR1 function (Hartmann et al., 2014; Muleya et al., 2016; Kaufmann et al., 2017).

Several genes related to PSK signal transduction have been identified. PSK treatment causes rapid protoplast expansion in wild-type Arabidopsis, whereas the cyclic nucleotide-gated channel 17 mutant is insensitive to PSK treatment (Ladwig et al., 2015). PSK transcription is regulated by RESPONSE FACTOR 115, which is involved in regulating cell division in the root quiescent center (QC; Heyman et al., 2013; Kong et al., 2018). RECEPTOR-LIKE PROTEIN 44 acts as a scaffold protein stabilizing the PSKR1–BAK1 complex. BRI1 regulates the transcription level of RECEPTOR-LIKE PROTEIN 44, thus influencing the interaction between PSKR1 and BAK1 (Holzwart et al., 2020; Garnelo Gomez et al., 2021).

PSK was originally identified as a peptide that can release the restriction of protoplast growth in liquid culture. Subsequent studies indicated that it is also involved in various aspects of plant growth, development, and immunity (Li et al., 2023b). For example, PSK can regulate cell division, differentiation, and expansion. Addition of PSK to liquid medium can induce protoplast expansion within minutes. The PSK signaling pathway has been shown to promote root and hypocotyl elongation by enhancing cell expansion rather than cell division in Arabidopsis thaliana (Kutschmar et al., 2009; Hartmann et al., 2013), and PSK has been reported to regulate seed size and yield in soybeans (Yu et al., 2019). PSK has also been found to promote nodulation in Medicago truncatula (Di et al., 2022; Yu et al., 2022) and somatic embryogenesis in Cunninghamia lanceolata (Hao et al., 2023).

PSK can coordinate plant growth and immune responses (Figure 1). In Arabidopsis, PSKR1 was found to inhibit the salicylic acid (SA) signaling pathway to limit the immune response while enhancing photosynthesis to promote growth (Song et al., 2023). A recent study revealed that PSK balances growth and defense responses in tomato by phosphorylating different amino acid residues of GLUTAMINE SYNTHETASE 2 (GS2) through CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28; Ding et al., 2023). The PSK pathway exhibits opposite functions in the immune response to biotrophic and necrotrophic pathogens (Figure 1). In response to biotrophic pathogens, the PSK pathway suppresses the immune response and promotes differentiation of host cells into specialized feeding cells. Conversely, in response to necrotrophic pathogens, the PSK signaling pathway can increase intracellular Ca²⁺ concentrations and activate an auxin-mediated immune response (Igarashi et al., 2012; Mosher and Kemmerling, 2013; Rodiuc et al., 2016; Zhang et al., 2018; Li et al., 2023b; Ding et al., 2023; Song et al., 2023). In this process, PSK inhibits the ubiquitination modification of its receptor by PUB12/13 (Figure 1), enhancing the stability of PSKR1 in tomato (Hu et al., 2023).

Figure 1.

A current model of the PSK signaling pathway.

PSK binds to its receptors, the PSKRs, causing recruitment of the BAK1/SERKs co-receptors to the complex and leading to the phosphorylation, guanylate cyclase and CaM binding activities of the PSKRs. These early events initiate a downstream signaling cascade that regulates plant cell division, cell expansion, immunity response and other critical processes. PSKR1/ALR1 serves as a sensor for monitoring intracellular aluminum ions.

PSK is involved in plant reproduction. Disrupting the PSK signaling pathway in Arabidopsis results in shorter pollen tubes, and PSK signaling is critical for guiding pollen tube growth into the embryo sac (Chen et al., 2000; Stuhrwohldt et al., 2015). In tomato, PSK triggers flower abscission under drought stress (Reichardt et al., 2020) and promotes fruit ripening by phosphorylating the transcription factor DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2F (DREB2F; Figure 1; Fang et al., 2024).

A recent study revealed that PSKR1, also named Al RESISTANCE 1 (ALR1) acts as a sensor for Al ions (Figure 1). The intracellular domain of PSKR1 can bind Al ions, inducing heterodimerization with the co-receptor BAK1. This leads to the generation of reactive oxygen species (ROS) via phosphorylating the NADPH oxidase RobhD. ROS, in turn, inhibit the ubiquitination-based degradation of the zinc-finger transcription factor SENSITIVE TO PROTON TOXICITY 1 (STOP1) through oxidative modification of the F-box protein REGULATION OF ATALMT 1 EXPRESSION 1 (RAE1; Ding et al., 2024). STOP1, which activates the secretion of organic acid anions to detoxify Al, is the key transcription factor for Al ion stress tolerance in plants (Iuchi et al., 2007; Liu et al., 2009). In summary, PSK and its receptors, PSKRs, play key roles in a number of biological processes essential for plant growth, development, and immunity. Future studies will focus on elucidating the detailed molecular mechanisms involved in these processes.

PSYs

The PSY peptides were originally isolated from Arabidopsis cell culture medium (Amano et al., 2007). Nine PSY genes were subsequently identified in the Arabidopsis genome. These genes encode PSY precursor proteins ranging from 71 to 104 amino acids in length (Amano et al., 2007; Tost et al., 2021; Ogawa-Ohnishi et al., 2022). Each precursor protein contains a signal peptide at the N terminus and a PSY core sequence at the C terminus. Other than that, there is no significant sequence similarity among the nine PSY precursor proteins. The mature PSY1 peptide consists of 18 amino acids, with a sulfation modification on Tyr2 and hydroxylation modifications on Pro16 and Pro17. The hydroxyl group of Pro16 carries three L-arabinose glycosyl residues. Some PSYs, however, lack these two post-translationally modified proline residues, leading to uncertainty about the effect of hydroxylation and arabinose glycosylation on the biological activity of the peptide (Kaufmann and Sauter, 2019; Tost et al., 2021).

Three closely related LRR-RLKs in the LRR XI subfamily were recently identified as the receptors of PSYs. The extracellular domains of all three LRR-RLKs can specifically interact with PSYs. These LRR-RLKs were subsequently named PSY RECEPTORs (PSYRs; Ogawa-Ohnishi et al., 2022). PSYRs were also called ROOT ELONGATION RECEPTOR KINASEs (REKs) in an earlier report because of their roles in negatively controlling root elongation in Arabidopsis (Wang et al., 2022). PSYRs can directly interact with PSYs. Unlike other well-studied ligands, such as PSK or brassinosteroids, which activate receptors upon binding, PSY1 actually inhibits the functionality of PSYRs (Figure 2; Ogawa-Ohnishi et al., 2022). Overexpression of PSYR1 results in a short-root phenotype, whereas addition of PSY1 to the medium or knockout of PSYRs/REKs leads to longer roots in Arabidopsis (Wang et al., 2022). An early report suggested that a member of the LRR RLK X subfamily called PSY1 RECEPTOR (PSY1R) is involved in PSY1 signaling (Amano et al., 2007). PSY1R can directly interact with and activate AHA2 by phosphorylating its T881 residue. PSY1 activates the plasma membrane H⁺-ATPase AHA2 in a PSY1R-dependent manner, leading to extracellular acidification and hypocotyl elongation (Amano et al., 2007; Fuglsang et al., 2014). Nonetheless, there is no evidence to demonstrate that PSY1R is the receptor of PSY1.

Figure 2.

A current model of the PSY signaling pathway.

PSYRs/REKs play a dual role by suppressing plant growth while stimulating stress responses. PSYs from Arabidopsis thaliana, RaxXs from Xanthomonas, and MigRSYs from Meloidogyne inhibit the functions of PSYRs through direct binding. PSYs have been reported to induce AHA2 activity in a PSY1R-dependent manner.

PSY1 regulates many different processes, including cell expansion and proliferation, cell wall development, epidermal development, and the balance between plant growth and stress responses (Amano et al., 2007; Mosher and Kemmerling, 2013; Shen and Diener, 2013; Ogawa-Ohnishi et al., 2022; Wang et al., 2022). In the long-standing battle between plants and pathogens, pathogens have evolved the ability to utilize the PSY signaling pathway to suppress plant defense responses. For example, Xanthomonas RaxX peptides are highly similar to PSY family peptides. RaxX16 or RaxX21 is able to bind to PSYRs to promote Arabidopsis root development, similar to PSY1 (Figure 2; Pruitt et al., 2017; Pruitt et al., 2015). In a recent study, Meloidogyne was found to secrete PSY-like peptides, called MigPSYs, to manipulate the plant immune system (Figure 2; Yimer et al., 2023). At the same time, plants have also evolved a mechanism to combat this problem. For instance, the rice receptor XA21 specifically recognizes RaxXs but not PSYs (Pruitt et al., 2015; Luu et al., 2019). These studies suggest that PSY regulation of immunity is a conserved process that has developed over a long period of coevolution. However, the detailed mechanisms regarding how PSY suppresses immunity remain unclear. These questions may be resolved through discovery of additional signal transduction elements and structural analysis of PSYRs.

RGFs

RGFs were identified independently by three laboratories using different strategies. Earlier studies indicated that the tpst mutant has a short-root phenotype, but addition of two known sulfated peptides, PSK and PSY1, to the culture medium did not completely rescue the short-root phenotype (Matsuzaki et al., 2010). This observation suggested that other unidentified sulfated peptides are able to regulate primary root development. Screening for these potential sulfated peptides led to the discovery of a new group of peptides named RGFs. Ten RGFs have been identified in the Arabidopsis genome (Matsuzaki et al., 2010; Shinohara, 2021). In a different study, CLE18 overexpression and in vitro CLE18 treatment led to different Arabidopsis root phenotypes (Meng et al., 2012). Sequence comparison revealed the presence of another peptide sequence, CLE LIKE (CLEL), in the middle of the CLE18 precursor protein. Sequence alignment revealed ten CLEL members in Arabidopsis. In an independent study, a new group of peptides were found to regulate root gravitropism. These eleven peptides were designated as GOLVENs (GLVs) (Whitford et al., 2012). RGFs, CLELs, and GLVs actually belong to the same peptide family, which contains a total of thirteen members. For convenience, this family of peptides is referred to as RGFs in this review article. The precursor proteins of RGFs are composed of 86–163 amino acid residues, including an N-terminal signal peptide, a conserved C-terminal sequence, and a variable sequence in the middle portion of the protein. RGF precursors can be cleaved into 13–18 amino acid residues and modified by sulfonation and hydroxylation to generate mature peptides. In RGF1, for example, Tyr2 is sulfonated and Pro10 is hydroxylated (Matsuzaki et al., 2010; Meng et al., 2012; Whitford et al., 2012; Shinohara, 2021).

RGFs are expressed in various organs and tissues of Arabidopsis. RGF1, RGF2, RGF3, RGF5, and RGF8 are expressed in the QC and the root caps; RGF4, RGF8, and GLV9 in the root meristem; RGF7 and GLV8 in the differentiation zone; RGF6 and RGF9 in the aerial tissues; and RGF1, RGF3, RGF5, RGF6, and RGF9 in the leaves (Fernandez et al., 2013).

The receptors of RGFs were independently identified by three research groups using different experimental approaches. Matsubayashi’s group from Nagoya University identified three LRR-RLKs as RGF receptors using a photoaffinity labeling approach. These three LRR-RLKs were named RGF RECEPTOR 1 (RGFR1), RGFR2, and RGFR3 and were demonstrated to interact directly with RGF1 (Shinohara et al., 2016). Li’s group from Lanzhou University used a yeast two-hybrid approach to identify five LRR-RLKs that could interact with BAK1. The quintuple mutant of these LRR-RLKs exhibited a short-root phenotype, similar to that of the bak1 serk1 double mutant. Because the quintuple mutant was completely insensitive to exogenous RGF1, the five LRR-RLKs were designated RGF1 INSENSITIVEs (RGIs; Ou et al., 2016). Chai’s and Guo’s groups from Tsinghua University and Beijing University, respectively, identified the same five LRR-RLKs using structural biology and motif analysis (Song et al., 2016). RGI1, 2, 3 correspond to RGFR1, 2, 3 (from Matsubayashi’s nomenclature), respectively. The structure of RGF1–RGI3 was solved. Arg458 and Arg460 of RGI3 interact with the free carboxyl group of the last residue, Asn13, of RGF1. The RxGG motif, including Arg195, Gly197, and Gly198 of RGI3, is highly conserved and recognizes the sulfate group of RGFs. In addition, Asp412 and Leu436 of RGI3 recognize Asn13 of RGF1 via hydrogen bonds and van der Waals forces. Trp390 of RGI3 interacts with the side chain of Asn13 of RGF1, and Asp174 of RGI3 forms hydrogen bonds and salt bridges with the amide nitrogen of Tyr2 and the free amine of Asp1 in RGF1 (Song et al., 2016). Subsequent studies revealed that BAK1 and its paralogs, the SERKs, act as co-receptors of RGI1 for sensing of RGF1 (Song et al., 2016; Ou et al., 2022). RGF1 can induce the heterodimerization of RGI1 with SERKs, leading to mutual phosphorylation. In the serk1 bak1 double mutant, the phosphorylation level of RGI1 remains unchanged even after treatment with RGF1. The size of the root tip meristem in the serk1 bak1 double mutant is also insensitive to RGF1 (Ou et al., 2022). In addition, RGF1 induces the phosphorylation and subsequent ubiquitination of RGI1. Two ubiquitin-specific proteases, UBP12 and UBP13, can maintain the stability of the RGI1 protein by deubiquitination (Figure 3D). The ubp12 ubp13 double mutant exhibits a short-root phenotype similar to that of the rgi1, 2, 3, 4, 5 quintuple mutant (An et al., 2018). Upon perception by the RGI1–SERKs complex on the plasma membrane, the RGF1 signal can be transduced to downstream regulatory components via a YODA–MKK4/5–MPK3/6 signaling cascade (Figure 3D; Lu et al., 2020; Shao et al., 2020).

Figure 3.

The multifunctional roles of the RGF signaling pathway in Arabidopsis.

(A) The RGF signaling pathway modulates plant immune responses.

(B) RGFs regulate lateral root development.

(C) The RGF1 pathway orchestrates the equilibrium between superoxide anions and hydrogen peroxide within the root apex by means of RITF1.

(D) RGF1 and its receptors, the RGIs, and PEP1 and its receptors, the PEPRs, regulate root tip growth and immunity in a pH-dependent manner. RGF1 also regulates root gravitropism.

The root meristematic zone continuously generates new cells for development of the entire root system. A small number of mitotically inactive cells in the meristematic zone are referred to as the QC. The QC and its single layer of surrounding stem cells form a structure called the stem cell niche (SCN), which is crucial for root system development. The RGF1–RGI1 signaling pathway maintains the SCN by regulating the transcript levels of PLETHORA 1 and PLETHORA 2 (PLT1, PLT2) and the stability of their encoded proteins (Matsuzaki et al., 2010; Ou et al., 2016; Shinohara et al., 2016). A high concentration of PLT1 and PLT2 proteins inhibits cell division, whereas a moderate concentration promotes cell division, and a low concentration induces cell differentiation. PLT1 and PLT2 form concentration gradients in the root meristem, with the highest levels in the QC. The plt1 plt2 double mutant exhibits a shorter primary root with a severely damaged SCN (Aida et al., 2004; Galinha et al., 2007). RGF1 is also able to induce the transcription of RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), which can enhance the stability of PLT2 by increasing the accumulation of superoxide anions in the meristematic zone (Figure 3D; Yamada et al., 2020). The balance between superoxide anions and hydrogen peroxide determines whether root tip cells maintain their division activity or undergo elongation (Figure 3C). Within the root apical meristem, cells primarily undergo lateral expansion while preserving their division capacity. In the elongation zone, however, cells are able to elongate, but their division activities are lost (Tsukagoshi et al., 2010). RGFs were also shown to regulate lateral root development in an MPK6-dependent manner (Fernandez et al., 2013, 2015; Fernandez and Beeckman, 2020). Auxin accumulates locally during lateral root initiation and development. In vitro treatments with GLVs/RGFs can markedly reduce lateral root formation by preventing auxin accumulation during lateral root initiation (Jourquin et al., 2023). PUCHI, a transcription factor that negatively regulates lateral root development (Hirota et al., 2007; Kang et al., 2013), is controlled by RGFs. The transcript level of PUCHI increases in roots upon RGF1 treatment (Figure 3B). Consistent with this finding, RGF1 treatment cannot alter lateral root numbers in the puchi-1 mutant (Jeon et al., 2023). RGFs were also found to regulate the activities of the auxin efflux proteins PIN3 and PIN7 (Jourquin et al., 2023). RGF1 plays a role in plant gravitropic responses (Figure 3D). RGI5 accumulates on the lower side of the lateral root cap after gravitropic stimulation, increasing auxin transport to the elongation zone by upregulating PIN2 phosphorylation (Xu et al., 2023). RGFs are able to regulate immune responses through multiple pathways (Figure 3A). RGF7 is induced by Pseudomonas syringae infection in Arabidopsis leaves. Increased levels of RGF7 can be sensed by RGI4 or RGI5, enhancing resistance to the pathogen (Wang et al., 2021; Stegmann et al., 2022). FLAGELLIN SENSITIVE 2 (FLS2) is a key receptor kinase in the regulation of plant immune responses (Gomez-Gomez and Boller, 2000). The RGF–RGI signaling pathway can help to maintain the abundance of FLS2, promoting the immune response (Stegmann et al., 2022). In Arabidopsis root tips, activation of either the RGF1–RGI1 or PEP1–PEPR signaling pathway depends largely on extracellular pH (Figure 3D). An acidic environment around the root tip promotes the binding of RGF1 to its receptor RGI1, facilitating growth and suppressing the immune response. By contrast, the RGF1 signaling pathway is inhibited when the root tip is in an alkaline environment, and the PEP1–PEPR-pathway-triggered immune response is enhanced (Liu et al., 2022).

Recent studies have revealed the mechanisms by which RGF1 regulates the root gravitropic response (Xu et al., 2023). Exogenous treatment with RGF1 was shown to upregulate the phosphorylation of PIN2. However, it remains unclear whether RGFs also induce the phosphorylation of other PINs like PIN3 and PIN7 during lateral root development (Jourquin et al., 2023; Xu et al., 2023). In addition, the transcript level of RITF1 is regulated by RGF1 during RGF signal transduction, but it is not yet clear whether this regulation occurs via the known YODA–MKK4/5–MPK3/6 pathway. Currently, our understanding of the functions of RITF1 is relatively limited. How RITF1 transcript levels are regulated and how superoxide anion accumulation is controlled by RITF1 need to be determined in the near future. Another key question is how RGF signaling is precisely regulated in cells. Both the absence and the hyperactivation of RGF signaling result in a short-root phenotype (Matsuzaki et al., 2010; Ou et al., 2016; Lu et al., 2020; Wang et al., 2021), suggesting that RGF signaling is strictly monitored. Identification of novel regulatory components, especially negative regulators, should help to resolve this fundamental issue.

CIFs and TWS1

Before the discovery of CIFs, TPST was found to regulate the development of the Casparian strip, operating in the same pathway as the two LRR-RLKs GASSHO 1 (GSO1) and GSO2 (Tsuwamoto et al., 2008). GSO1 is also known as SCHENGEN 3 (SNG3 Pfister et al., 2014). However, none of the previously discovered sulfated peptides were found to participate in Casparian strip development. This led to the discovery of a new group of peptides that could bind directly to GSO1 and GSO2, known as CIFs, including CIF1 to CIF4 (Doblas et al., 2017; Nakayama et al., 2017). Both the tpst single mutant and the gso1 gso2 double mutant exhibit seed twisting and defective cuticle permeability in addition to Casparian strip defects (Creff et al., 2019; Doll et al., 2020). However, the cif1, 2, 3, 4 quadruple mutant did not show similar defective phenotypes, suggesting the existence of other unidentified CIF peptides (Doll et al., 2020). TWISTED SEED 1 (TWS1) was initially identified as a small protein containing 81 amino acids. The tws1 mutant shows distorted seeds and defective cuticle deposition (Fiume et al., 2016). Sequence analysis revealed the presence of a CIF structural domain in the middle of the TWS1 protein. Subsequent experiments confirmed that TWS1 is in fact a CIF-like peptide. It serves as one of the ligands of GSO1 and GSO2, participating in seed and cuticle development (Doll et al., 2020). Similar to other sulfated peptides, the CIFs and TWS1 must be cleaved from their precursor proteins and modified to become mature peptides. For example, the mature CF1 peptide consists of 21 amino acids with sulfation at Tyr2 and hydroxylation at Pro7 and Pro9. The core sequences of CIF1 and CIF2 are located at the C termini of their precursor proteins. Although the core sequences of CIF3, CIF4, and TWS1 are also at the C termini of their precursor proteins, several amino acid residues must be cleaved before they reach full activity (Nakayama et al., 2017; Doll et al., 2020).

CIFs and TWS1 regulate the development of the root Casparian strip, embryo epidermal layer, and pollen cell wall (Figure 4). Unlike other sulfated peptides, CIFs and TWS1 exhibit diverse functions throughout various developmental processes. The Casparian strip, located in the cell wall of the endodermis, is a barrier that regulates the diffusion of water and mineral elements in plants. During development of the Casparian strip, CIF1 and CIF2 are expressed in the stele, whereas GSO1 is localized on both sides of the Casparian strip formation site (Doblas et al., 2017; Nakayama et al., 2017; Fujita et al., 2020; Zhang et al., 2024). The signal transduction pathway also relies on the cytoplasmic receptor kinase, SCHENGEN 1 (SGN1), which is localized on the inner side of the Casparian strip formation site near the endodermis (Doblas et al., 2017; Fujita et al., 2020). After passing through the newly formed but not yet closed Casparian strip, mature CIF1 or CIF2 is perceived by its receptor GSO1 located on the cortical side. The signal is then transduced through SGN1, promoting formation of a complete Casparian strip (Figure 4A). A fully developed Casparian strip can prevent further movement of CIF1 or CIF2 peptides into the endodermal tissue, thereby terminating signal transmission (Doblas et al., 2017; Nakayama et al., 2017; Fujita et al., 2020). CIF3 and CIF4 play a crucial role in regulating pollen development (Truskina et al., 2022). Pollen development takes place within the anther locules, which are surrounded by four somatic cell layers: the epidermis, endothecium, middle cell layer, and tapetum (from outside to inside). CIF3 and CIF4 are expressed in the tapetum layer. CIF3 and CIF4 precursor proteins require cleavage by the protease SBT5.4. SBT5.4 is expressed in pollen. After being secreted into the extracellular space, SBT5.4 can pass through the incompletely closed pollen cell wall, processing the CIF3 and CIF4 precursor proteins into mature peptides. Mature CIF3 or CIF4 can be sensed by GSO1/GSO2, which are expressed in the middle layer, stimulating formation of the pollen wall (Figure 4B). Complete closure of the pollen wall prevents the passage of SBT5.4, thereby interrupting activation of the CIF3 and CIF4 signals (Truskina et al., 2022). The integrity of the embryonic cuticle, a physical barrier that covers the surface of the cell wall, is critical for separating the embryo from the neighboring endosperm. TWS1 was found to participate in development of the embryonic cuticle (Fiume et al., 2016). Both TWS1 and GSO1/GSO2 are expressed in the embryo (Tanaka et al., 2001; Yang et al., 2008; Creff et al., 2019). After expression in the embryo, the TWS1 precursor moves to the endosperm, where it is processed by the serine protease ABNORMAL LEAF-SHAPE 1 (ALE1) to form a mature peptide. Mature TWS1 then re-enters the embryo and is recognized by the receptors GSO1/GSO2 and co-receptors BAK1/SERKs (Zhang et al., 2022), stimulating formation of the cuticle barrier (Figure 4C). Complete closure of the cuticle barrier prevents TWS1 from moving to the endosperm side, thus blocking continued activation of the signaling pathway (Doll et al., 2020). GSO1 and GSO2 also play a crucial role in plant responses to salt stress (Chen et al., 2023). They contribute to salt-stress tolerance by maintaining the integrity of the Casparian strip, which prevents diffusion of Na⁺ into the vascular system and thereby reduces potential damage to the plant. GSO1 has been shown to interact with and activate SALT OVERLY SENSITIVE 2 via phosphorylation (Chen et al., 2023). SALT OVERLY SENSITIVE 2 can prevent accumulation of Na⁺ inside cells (Liu et al., 2000; Lin et al., 2009; Li et al., 2023a), further aiding plants in coping with salt stress. Previous studies have shown that the CIF/TWS1 signaling pathway is involved in regulating the formation of multiple barriers in plants (Chen et al., 2023). The detailed molecular mechanisms by which the CIFs and TWS1–GSO1/GSO2 control various barriers remain unclear. In addition, how the functions of CIFs and TWS1 are diversified is another interesting topic for future exploration.

Figure 4.

A current model of the CIFs and TWS1 signaling pathway.

(A) CIF1 and CIF2 and their receptors, GSO1/GSO2, are required for formation of the Casparian strip.

(B) CIF3 and CIF4 and their receptors, GSO1/GSO2, regulate pollen wall development.

(C) TWS1 and its receptors, GSO1/GSO2, are involved in closure of the embryonic cuticle during embryo development.

Concluding remarks and perspectives

Sulfated peptides play essential roles in various biological processes during the plant life span, helping to balance plant growth and stress adaptation. For example, PSKs coordinate plant growth and immune response (Ding et al., 2023; Song et al., 2023), RGF1 is involved in pH-dependent root tip growth (Liu et al., 2022), and PSYs are crucial for maintaining the equilibrium of root development and stress responses (Ogawa-Ohnishi et al., 2022). In addition, sulfated peptides have been shown to contribute to prolonging the shelf life of fruits and vegetables. Application of RGF1 can extend the storage time of peaches (Tadiello et al., 2016), and PSK treatment can prolong the storage time of broccoli and strawberries (Aghdam et al., 2021; Aghdam and Flores, 2021; Aghdam et al., 2020). More detailed roles of the sulfated peptides are shown in Supplemental Table 1. These observations suggest that sulfated peptides have potential commercial applications in agriculture.

Balancing plant growth and stress tolerance through artificial intervention is one of the purposes of plant biology. Traditional hormones typically have broad regulatory effects on plant growth and development, making it difficult to intervene in only one specific process. By contrast, sulfated peptides have relatively more specific effects on plants, making them more suitable for agricultural application. Moreover, exogenous spraying of low concentrations of sulfated peptides has been reported to activate plant responses. However, de novo chemical synthesis of sulfated peptides is too costly, limiting their application in agriculture. The discovery of bacterial RaxX peptides suggests the exciting possibility of large-scale, low-cost sulfated peptide production using biological reactors.

Although many studies have confirmed the importance of sulfated peptides, existing research has focused predominantly on their functions and receptors. Many questions still remain unanswered. In the future, research on sulfated peptides will shift toward revealing how their signals are transmitted and regulated at the cellular level. With the help of traditional genetics and newly developed technologies such as proximity labeling, proteomics, and phosphoproteomics, there is hope for accelerating research on the molecular mechanisms of sulfated peptide signal transduction in the near future.

Supporting citations

Fernandez et al., 2020; Ghorbani et al., 2016; Matsubayashi and Sakagami, 1998; Matsubayashi et al., 1997; Matsubayashi et al., 1999; Motose et al., 2009; Stuhrwohldt et al., 2021; Yang et al., 1999; Yu et al., 2016.

Funding

Research in the authors’ lab is currently supported by the National Natural Science Foundation of China (no. 32030005).

Author contributions

J.L. and L.H. conceived the review topic. L.H. and L.W. prepared the draft. J.L. and L.H. revised the manuscript.

Published: April 10, 2024