Plant peptides – redefining an area of ribosomally synthesized and post-translationally modified peptides

Jonathan R. Chekan *^a, Lisa S. Mydy ^b, Michael A. Pasquale ^a and Roland D. Kersten *^b
aDepartment of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA. E-mail: jrchekan@uncg.edu
^bDepartment of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA. E-mail: rkersten@umich.edu

First published on 27th February 2024

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Jonathan R. Chekan

Jonathan R. Chekan is an Assistant Professor of Chemistry and Biochemistry at the University of North Carolina at Greensboro. He received his PhD in 2016 from the University of Illinois Urbana-Champaign under the supervision of Satish K. Nair. As the Simons Foundation Post-Doctoral Fellow of the Life Science Research Foundation in the laboratory of Bradley S. Moore, he investigated the biosynthesis of neurotoxic marine natural products. In 2020, he joined University of North Carolina at Greensboro where his research group focuses on the bioinformatic guided discovery of new natural product biosynthetic pathways from across the tree of life.

Lisa S. Mydy

Lisa Mydy is fascinated by protein structure and function in natural product biosynthesis. She earned her PhD in Chemistry with Dr Nicholas Silvaggi from the University of Wisconsin-Milwaukee in 2016. She was a postdoc with Dr Andrew Gulick at the University at Buffalo, spent time in industry with Abbott, and is currently a postdoc in Dr Roland Kersten's laboratory at the University of Michigan. She determined the first crystal structure of a BURP domain protein with the co-mentorship of Dr Janet Smith and plans to continue exploring the BURP superfamily.

Michael A. Pasquale

Michael A. Pasquale is pursuing his PhD in chemistry and biochemistry at the University of North Carolina at Greensboro. He is in the lab of Jonathan R. Chekan and his research focus is the biosynthesis of RiPP natural products in plants. Prior to his graduate studies, in 2021 Michael received his B.S. in Biochemistry from the University of North Carolina at Greensboro, where he studied natural product chemistry with Dr Nadja Cech.

Roland D. Kersten

Roland Kersten is an Assistant Professor of Medicinal Chemistry at the University of Michigan, Ann Arbor. He pursued graduate studies in Dr Pieter Dorrestein's and Dr Brad Moore's labs at University of California, San Diego on microbial genome mining for natural product discovery. He then was trained as a postdoc in Dr Joseph Noel's and Dr Jing-Ke Weng's labs at Salk Institute and Whitehead Institute in plant and algal natural product biosynthesis. Roland's lab develops gene-guided discovery approaches for plant natural products.

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1. Introduction

RiPPs are a large, diverse group of natural products which are produced by bacteria, archaea, fungi, animals, and plants. The defining RiPP biosynthetic feature is the ribosomal generation of a precursor peptide which is post-translationally modified (PTM) and proteolytically cleaved to yield a mature RiPP natural product^1,2 (Fig. 1A). Plants are a prolific source of peptide chemistry³ and the investigation of underlying peptide biosynthetic genes has revealed that plants produce peptide natural products exclusively via the ribosomal pathway based on current knowledge.^4–21 Plant RiPPs have yielded sustainable pest control agents in agriculture such as Sero-X,^22,23 and they have emerged as promising lead compounds for medicinal applications exemplified by the clinical lead immunosuppressant T20K.²⁴ With growing plant genetic resources²⁵ and recent advances in identification of precursor peptides and biosynthetic enzymes involved in plant RiPP biosynthesis, the field of plant RiPPs is primed for an expansion of plant RiPP chemistry and enzymology that will further advance research about their agricultural and therapeutic applications. This review aims to give an overview of plant RiPP chemistry, biosynthesis, and bioactivity while also recommending nomenclature for the recent discovery of side-chain-macrocyclic RiPPs produced by BURP-domain peptide cyclases.

In this review, we will first introduce plant peptide chemistry to highlight characterized ribosomal peptides with post-translational modifications. We will then give insights into plant RiPP biosynthetic trends based on current biochemical knowledge (Section 2). Next, plant RiPP classes will be reviewed in structure, biosynthesis, and bioactivity (Section 3). We summarize the distribution of these plant RiPPs across vascular plants in Section 5. Finally, discovery approaches for plant RiPPs are described (Section 6) and we look ahead in future research directions of plant RiPPs (Section 7). We also review biosynthetically undefined peptides derived from plants (Section 4). This review has a particular focus on BURP-domain-derived RiPPs due to their recent biosynthetic discovery. For the other plant peptide classes, we further recommend excellent reviews discussed in their respective sections.

2. Plant peptide chemistry and biosynthesis

To date, over 1500 modified peptides have been isolated from plants.^3,18,26,27 Detailed biosynthetic studies have firmly established the biosynthetic basis for many of these peptides, allowing for classification based on both structure and construction (Fig. 1B). The plant itself is typically responsible for biosynthesis of the RiPP, which is contrary with other sessile organisms like sponges that rely on a complex microbiome to produce the observed bioactive metabolite.^28–30 There are notable exceptions to this trend in plants, such as the macrocyclic depsipeptide FR900359,³¹ which is produced by the endosymbiotic bacterium Candidatus Burkholderia crenata in the leaves of the plant Ardisia crenata. FR900359 is a potent inhibitor of the Gq subfamily of G protein signaling, a novel therapeutic target for treating asthma, inflammation, and cancer.^32–35 Other peptidic natural products derived from plant sources such as the bouvardins still await biosynthetic classification (Fig. 1B and Section 4).

Plant RiPPs have a wide range in size, from four amino acids (cyclopeptide alkaloids)³⁶ up to 66 amino acids (cysteine-rich peptides)³⁷ or 430 Da to 7 kDa (Fig. 1B). A common structural feature of plant peptide natural products is macrocyclization. Macrocyclic plant RiPPs can be constructed in a variety of ways: head-to-tail (cyclotide), side-chain-to-side-chain (cyclopeptide alkaloids) or side-chain-to-backbone (lyciumins). Below we describe important features of plant RiPP pathways following general RiPP biosynthetic order of precursor peptide formation, post-translational modification, and proteolysis.

2.1. Precursor peptides

As characteristic of RiPPs, genetically encoded precursor peptides serve as the substrate for the biosynthetic pathways. Precursor peptide sequences can often be subdivided by their functional role. Since many contrasting definitions have been used for plant precursor peptide organization over the past decades, we propose the following in addition to accepted RiPP precursor peptide definitions:^1,2

• Leader (peptide) is the non-repeating N-terminal portion of the precursor peptide following the signal sequence (if a signal peptide is present) before the first N-terminal core peptide.

• Core (peptide) is a sequence of the precursor peptide which is transformed to the RiPP natural product.

• Recognition sequence is the repeating unit demarcating core sequences in multi-core precursor peptides.

• Follower (peptide) is the non-repeating C-terminal portion of the peptide following the core sequence.

Leader, follower and recognition sequences often function as regions for interactions with PTM enzymes or domains and proteases in RiPP pathways.^1,2 The presence of leader peptide, follower peptides, or recognition sequences is not a requirement for plant RiPP precursor peptides.

Plant RiPPs can be derived from single-core and multi-core precursor peptides (Fig. 1C). Most plant RiPP classes have precursor peptides that have been characterized to include more than one core peptide in sequence repeats, known as multi-core precursor peptides (Table 1).³⁸ For example, two of the originally reported cyclotide precursor peptides (Oak2, Oak4) from Oldenlandia affinis include 2 or 3 core peptides, respectively, but precursor peptides with only one cyclotide core peptide (Oak1, Oak3) were reported from the same plant.⁴ Other multi-core precursor peptides have been described for orbitides,³⁹ linear RiPPs,¹¹ several cysteine-rich peptides,^40–42 lyciumins,¹⁷ moroidins¹⁹ and cyclopeptide alkaloids^18,20 (Table 1). Multi-core precursor peptides are also in biosynthetic pathways of fungal RiPPs such as dikaritins^43–46 and borosins,^47,48 and cyanobacterial RiPPs such as cyanobactins^49,50 and microviridins.⁵¹ A multi-core precursor in plant RiPPs could have the advantage over a single-core precursor in that RiPP production could be increased faster and more efficient, i.e. transcription and translation of one precursor gene can yield multiple plant RiPP products. The evolution of multi-core precursors has been hypothesized to occur through internal gene duplication and neofunctionalization in the form of core peptide diversification, as observed for squash trypsin inhibitor cyclotides.⁵ Many precursor peptides from plant RiPP pathways are above 100 amino acids in length (Table 1 and Fig. 1D) and therefore larger than typical precursor peptides from bacterial RiPPs.^1,2 The large size of plant precursor peptides arises for several reasons: (a) plant RiPP precursor peptides often include signal peptides for processing through the secretory pathway in plant cells, (b) they can include multiple core peptides,^4,5,17 (c) core peptides have evolved into storage proteins^22,52,53 and (d) precursor peptides can be fused to catalytic domains.^18,54 In some cases, the large size of plant RiPP precursors has been hypothesized to be a result of cyclic peptide motifs ‘hijacking’ plant proteins and their processing enzymes for biosynthesis.⁵² Such cyclic peptide motifs can function as stand-alone inhibitor protein loops like in Bowman–Birk-inhibitors⁵⁵ or serve as RiPP core peptides for post-translational modification and proteolysis during cyclic peptide biosynthesis.⁶

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2.2. Post-translational modification

Bacterial RiPPs are often heavily modified to contain many non-proteogenic amino acids, whereas plant RiPPs are generally comprised of proteinogenic amino acids with only a limited number of post-translationally modified amino acids. Plant RiPP PTMs can be differentiated into terminal modifications such as N-to-C-macrocyclization or pyroglutamate formation and into side-chain-modifications such as crosslinking of tryptophan and tyrosine side chains with other amino acids (Fig. 2). Multiple plant RiPP classes feature PTMs common to eukaryotic proteins, including cystines, pyroglutamate, tyrosine sulfation, and proline hydroxylation.⁵⁶ We rationalize the inclusion of common protein PTMs in plant RiPPs because of the general definition of RiPPs as ribosomal peptides with a post-translational modification and an altered structure–activity-relationship (e.g. a change in biological target affinity, stability, conformation, charge state) of the unmodified ribosomal peptide through the PTM. For example, cystines were not detailed in seminal RiPP reviews as PTMs defining a ribosomal peptide as post-translationally modified, but one of these reviews mentions ribosomal peptides with disulfide bonds, so called cysteine-rich peptides (CRPs), following RiPP biosynthetic logic.^1,2 We suggest including cystines as RiPP-defining PTMs given their nature as a covalent precursor peptide modification and their impact on peptide SAR (Section 3.4). Plant RiPPs with cystines are cyclotides, PawS-derived peptides, and CRPs. In contrast to established RiPP-defining PTMs which are only formed enzymatically, cystines can form non-enzymatically through oxidative folding^57,58 or enzymatically by protein disulfide isomerases.⁵⁹

Plant RiPP biosynthesis can occur through a split pathway or an autocatalytic (fused) pathway (Fig. 1E). Most plant RiPPs such as cyclotides, orbitides, PawS-derived peptides and cyclopeptide alkaloids are derived from precursor peptides, which are expressed as separate proteins from their PTM enzymes (Table 1). Recently, several BURP-domain-containing proteins (Section 3.6) have been characterized which can catalyze macrocyclic PTMs in core peptides within the same protein as the cyclase domain. This discovery showed that plant RiPP biosynthesis can involve autocatalysis,¹⁸ a biochemical mechanism first observed for peptide-N-methyltransferases of fungal RiPP pathways in borosin biosynthesis.^60,61

2.3. Proteolysis

Plant RiPP biosynthesis utilizes protein processing enzymes found in secretory pathways. For example, the biosynthesis of cyclotides involves asparaginyl endopeptidases (AEP), proteolytic enzymes often involved in processing vacuolar proteins like vacuolar storage protein albumins.^62,63 Precursor peptides with albumin domains are indeed observed for head-to-tail-macrocyclic plant RiPPs PawS-derived peptides, PawL-type orbitides, and cyclotides. The genetic, structural, and biochemical connection of plant RiPP chemistry to larger plant proteins indicates evolutionary roots of some plant RiPPs from functional motifs in such proteins.^52,55

3. Plant RiPP classes

3.1. Cyclotides

Cyclotides are defined by a head-to-tail-macrocyclization and the presence of a cystine knot motif in their structure (Fig. 3). The founding member kalata B1 was discovered as an oxytocic agent in extracts of the Congolese plant Oldenlandia affinis, which is used as an herbal medicine to facilitate child birth during labor.^80–83 Since the discovery of O. affinis cyclotides, many other cyclotides have been characterized with 761 cyclotides documented in the cyclic plant peptide database CyBase^26,84 that establishes cyclotides as the largest plant RiPP family to date. For more details about cyclotides, we direct readers to several dedicated reviews.^85,86

3.2. Orbitides

Orbitides are small head-to-tail-macrocyclic RiPPs with no disulfide bond (Fig. 4). They were originally described as Caryophyllaceae-type cyclic peptides because some founding members of these compounds, such as nonapeptide cyclolinopeptide A from flax (Linum usitatissimum), were discovered from plants in the pink family.¹²¹ A wider taxonomic distribution of these compounds was revealed in the same and following decades^122–124 and the precursor genes of several orbitides were sequenced and confirmed recently which defined them as RiPPs.^{6,7,39,77,123,125,126} In 2013, the name orbitides was proposed for these head-to-tail-cyclic RiPPs without disulfide bonds.¹ To date, nearly 200 orbitides have been deposited to CyBase^26,84 and are broadly distributed across plants as they are found in the Asterales, Caryophyllales, Lamiales, Magnoliales, Malpighiales, and Sapindales orders.

3.3. PawS-derived peptides

PawS-derived peptides (PDPs) are head-to-tail-macrocyclic RiPPs with one disulfide bond. The founding member of the PDPs, sunflower trypsin inhibitor (SFTI-1 Fig. 5), was discovered in 1999 from sunflower seeds (Helianthus annus).¹⁵² Additional isolation and genetic mining efforts have revealed around 20 total PDPs.^52,124,153 These natural products do not appear to be widespread in plants and are largely localized around species phylogenetically related to H. annus.¹²⁴ We recommend a review that focuses on SFTI-1 with many details on the extensive engineering efforts of SFTI-1¹⁵⁴ and another review that discusses PDPs more broadly⁵³ for further reading.

3.4. Cysteine-rich peptides

In addition to cyclotides, plants produce many peptides without head-to-tail-macrocyclization, but with multiple cysteines that engage in macrocyclization via disulfide bonds. The classification of cysteine-rich peptides (CRPs) is historically based on activity (antimicrobial peptides, defensins, rapid alkalinization factors), structure (knottins, thionins, snakins), or chemotaxonomy of founding members (heveins). Given current knowledge of cysteine-rich peptide (CRP) chemistry and biosynthesis, the most applicable classification scheme for CRPs is the number and pattern of disulfide bonds, which translates into distinct structures (Fig. 6). CRP precursor peptide sequences can differ significantly in size and domain structure within individual classes so that core peptide sequence and its cysteine pattern appear to be the best features for CRP prediction (Fig. 6). In addition, the net charge of a core peptide sequence is often considered for CRP characterization (Fig. 6A). Multiple excellent reviews have been published for CRPs.^162–164

3.5. Linear plant RiPPs

In addition to cyclic RiPPs, multiple linear plant peptides with post-translational modifications are produced by plants. Many of these linear RiPPs have roles in plant development and growth and are often secreted. The most common post-translational modifications in linear plant RiPPs are known from protein biosynthesis:⁵⁶ tyrosine sulfation,²⁹⁶ proline hydroxylation, and hydroxyproline arabinosylation (Fig. 2).²⁹⁷ We direct readers to dedicated reviews for additional details^296,298,299 as we discuss them only briefly.

3.6. BURP-domain-derived RiPPs (burpitides)

BURP-domain-containing proteins were characterized as precursor peptides for lyciumins and therefore for plant RiPP biosynthesis in 2018.¹⁷ Subsequently, several additional RiPP classes with new macrocyclic PTMs were revealed as BURP-domain-derived peptides (Fig. 8).^18–20 Given the rapid expansion of new RiPP classes derived from the BURP domain,³¹³ we propose the name of burpitide for this family of peptide natural products biosynthesized using a BURP-domain-containing protein.

The BURP-domain-containing protein family was defined by Hattori and co-workers based on a conserved CHX₁₀CHX_25–27CHX_25–26CH-motif³¹³ in four founding member plant proteins: microspore-derived embryo protein BNM2 from Brassica napus,³¹⁴ an unidentified seed protein (USP) from Vicia faba,³¹⁵ drought-responsive protein RD22 from Arabidopsis thaliana³¹⁶ and the β-subunit of polygalacturonase 1 (PG1β) involved in fruit ripening in Solanum lycopersicum.³¹⁷ Bioinformatic analyses of plant genomes show an abundance of BURP-domain genes with diverse primary structures and on average twelve BURP-domain genes per plant genome, ranging from one (Marchantia polymorpha) to 53 (Coffea arabica).³¹⁸ BURP-domain protein expression is mainly associated with abiotic stress responses in plants.^319,320 Examples are the expression of Sali3-2 in soybean roots during acidic soil stress.³¹⁹ Some BURP domains are potentially involved in biotic plant stress responses, for instance, a BURP-domain gene is up-regulated in a virus-resistant soybean³²¹ and a BURP-domain gene locus mapped to bruchid resistance in mung beans.³²²

In 2022, several BURP-domain-containing precursor peptides were reported as copper-dependent peptide cyclases, which catalyze macrocyclic bond formations in RiPP biosynthesis and define the autocatalytic or fused burpitide pathway.¹⁸ In 2023, a BURP-domain peptide cyclase with a stand-alone precursor peptide was reported defining the split burpitide pathway.²⁰

4. Undefined plant-derived peptides

While the molecules discussed in Section 3 have been established as plant produced RiPP natural products, numerous other peptides have been isolated from plants for which little biosynthetic information is available. Moreover, the authentic producer may also be unclear. In many cases, plants harbor endophytic bacteria or fungi that are associated with a particular species or genus. These microorganisms have been shown to be responsible for the production of many peptidic natural products, further complicating biosynthetic elucidation of a target molecule.^31,360 For example, the astin class of peptides was shown to be biosynthesized by an endophytic fungus.³⁶¹ Below we discuss one prevalent class of biosynthetically undefined bioactive plant peptides, the bouvardins.

4.1. Bouvardins

Bouvardins are a particularly noteworthy class of cyclic peptides isolated from plants for which both the true producer and biosynthetic route is unknown. Bouvardin (Fig. 12) was first isolated in 1977 from Bouvardia ternifolia.³⁶² Since then, over 30 analogues have been found in members of the Rubia genus including the RA-series,^363,364 the rubiyunnanins,^365,366 and the rubicordins.³⁶⁷

5. Chemotaxonomy of plant peptides

We sought to understand the chemotaxonomic distribution of plant RiPPs to identify trends and hot spots where specific peptides may be found. To assemble this data, we identified the order responsible for producing every known molecule in the cyclotide, PawS-derived peptide, orbitide, cysteine rich peptide, and burpitide classes of RiPPs. The presence of these peptides was limited to vascularized plants (Tracheophyta). To visualize the distribution of the plant RiPPs in tracheophytes, we collected the orders within Tracheophyta from the NCBI taxonomy database and used a combination of phyloT and the ggtree R package to generate a cladogram.³⁷⁶ The cladogram was then annotated with the RiPP classes data. Our cladogram does not include RALF peptides.

Fig. 13A shows the presence of the different RiPP classes found in plants. The results indicate that eudicots are the most prolific producers of RiPP natural products in plants, with representatives of all five types present. In particular, the rosid clade, including members such as Fabales and Malpighiales, appears to be a particular biosynthetic hot spot. Also of note is the Asterales order which has been shown to biosynthesize all five classes of plant RiPPs. Outside of eudicots, monocots also contain members that produce RiPPs, but to a lesser extent. The true ferns (Polyopsida) have had no RiPPs from these five classes identified. The cladogram also revealed a differential distribution of each plant RiPP. For example, PawS-derived peptides (PDPs) have only been found in the order Asterales. In contrast, cysteine-rich peptides and burpitides are more widely distributed across nearly all Tracheophyta clades.

We also generated a second cladogram focused on the distribution of the burpitides (Fig. 13B). Burpitide diversity seems to follow the same trend as all plant RiPP classes because the rosids have the largest diversity of isolated burpitides. Specifically, the Fabales and Rosales both contain three out of the four types of molecules contained within the burpitide class. This cladogram also highlights the fact that cyclopeptide alkaloids are the most widely distributed burpitide discovered to date.

It is important to note that while this figure may not show the authentic distribution of plant RiPPs in nature. As with all isolation experiments, the results can be biased by what specific molecules are being sought, what plant materials are available, and the isolation methodology. The increasing prevalence of plant genomes and transcriptomes is offering a way to target isolation in unexpected producers, such as selanine A in the African clubmoss of the Selaginellales order (see Section 3.6.3).

6. Discovery of plant RiPPs

Plant RiPPs have been discovered through bioactivity-guided, structure-guided, and gene-guided approaches that will be reviewed based on representative examples rather than a comprehensive list of discoveries below. A bioactivity-guided approach is less selective for compound classes, whereas structure-guided and gene-guided approaches can be effective in targeted discovery of RiPPs from plants.

6.1. Bioactivity-guided discovery

Multiple plant RiPPs have been discovered from source plants through their bioactivity as described in the respective bioactivity sections of Section 3. The choice of source plants is often inspired by application of its extracts in herbal medicine such as kalata B1 as an oxytocic agent, stimulation of pain such as excelsatoxin A as Na_V channel modulator, antimicrobial activity against plant pathogens such as α/β-purothionins, or plant physiological activities such as RALFs.

Challenges of bioactivity-guided discovery can be time-consuming fractionation and rediscovery of known bioactive natural products. To this end, a pipeline called PepSAVI (statistically-guided bioactive peptides prioritized via mass spectrometry) was developed to correlate bioactivity of pre-fractionated plant extracts with the presence of peptide mass signals in liquid-chromatography mass spectrometry datasets of the extracts. The masses of candidate bioactive peptides identified by this pipeline enable dereplication by MS1 and tandem MS approaches that was validated by the characterization of cyclotide cyO2 as an antibacterial and cancer cell cytotoxic lead compound from a Viola odorata extract fraction library.³⁷⁷ The assay further enabled discovery of candidate bioactive cyclotides from V. odorata by improved tandem mass spectrometry-based sequencing of target peptides via ultraviolet photodissociation (UVPD) tandem mass spectrometry.³⁷⁸ Pipelines such as PepSAVI that connect high-throughput bioactivity assay data with mass spectrometry can rapidly dereplicate and identify new bioactive plant RiPPs.³⁷⁹ MS-based approaches such as molecular networking³⁸⁰ or Dereplicator+³⁸¹ can enable rapid dereplication³⁸² and discovery³⁸³ of bioactive lead compounds via spectral library comparison, for example within the Global Natural Product Molecular Networking platform,³⁸⁴ and they can further assess structural novelty by spectral library comparison³⁸⁵ or de novo structure elucidation.^386,387

6.2. Structure-guided discovery

Many plant RiPPs were discovered based on physicochemical properties during natural product isolation or their structural features revealed by analytical chemistry techniques such as mass spectrometry (MS) as an initial chemotyping step.

Heveins were isolated from a centrifugation fraction including lutoids, vacuoles in latex-producing cells of rubber tree,³⁸⁸ and cliotides were discovered from heat stable extract fractions as compounds with high heat stability, a well characterized feature of cyclotides and CRPs.³⁸⁹ Besides physicochemical features, plant RiPPs have been effectively characterized via mass spectrometry approaches. Structural information can be derived from a peptide analyte via tandem mass spectrometry experiments; however it can be challenging as many plant RiPPs have macrocyclic PTMs, which often prevents sequencing of a given peptide. Therefore, cysteine-rich peptides usually require disulfide bond reduction and S-alkylation before tandem mass spectrometry analysis to characterize the complete core peptide sequence, analogous to proteomic protocols for MS-based protein sequencing.^390–392 The characteristic mass shift of iodoacetamide-alkylation at reduced cysteines (+57.0215 m/z) has been used for discovery of disulfide-containing plant RiPPs in plant extracts.^{40,65,71,173,175,184,252,389} A plant extract can be analyzed for the presence of analytes in the mass range of CRPs and cyclotides (2–6 kDa), treated for disulfide reduction and cysteine alkylation, then analyzed for alkylation-specific mass shifts in candidate peptides by comparison to untreated samples. For cyclotides, an additional step of proteolysis is usually required after disulfide reduction and cysteine alkylation to linearize the head-to-tail-macrocycle prior to tandem MS analysis. Proteolysis often involves GluC due to the presence of a conserved Glu in cyclotide core peptides²² or trypsin which can yield multiple tryptic core peptide species.⁹⁷ Despite the efficacy of chemical and proteolytic processing of cyclotides and CRPs prior to tandem MS sequencing, the main disadvantage of this sample preparation is higher sample requirement, which can exclude low abundance peptides from discovery. Recently, ultraviolet photodissociation (UVPD) tandem mass spectrometry has been introduced to yield more fragmentation and therefore peptide sequencing data from cyclotides and CRPs in underivatized plant extract samples.³⁷⁸ In addition, collision-induced dissociation, which is the predominant tandem MS method in metabolomics, was further developed for MS fingerprinting via short sequence tags enabling cyclotide dereplication and prioritization for discovery.³⁹³ For burpitides, tandem mass spectrometry (MS/MS) can be applied for characterization of structural features to reveal the presence of a peptidic analyte and specific amino acids within the peptide sequence.^18,394 In addition, tandem mass spectral data can be used in comparison to spectra of known plant peptides in custom or public databases for peptide classification.^{384,395–397} All aspects of peptide identification via MS/MS can inform novelty of a putative plant peptide and guide further experiments. Predicted amino acids and the size of the target analyte can enable the connection of a core peptide in a BURP-domain precursor peptide to the target spectrum. This approach has been applied effectively for the discovery of lyciumin-type and stephanotic acid-type burpitides, and cyclopeptide alkaloids. It can also be applied without a precursor peptide sequence as exemplified by the discovery of the glycosylated cyclopeptide alkaloid encephanine.¹⁸ The connection of tandem mass spectra of burpitides to their corresponding precursor peptides sequences has been implemented in the RiPP discovery platform HypoRiPPAtlas, which enables prediction of RiPP structures from candidate core peptide sequences in BURP-domain-containing precursor peptides. Subsequently, an algorithm called Dereplicator+³⁸¹ is used to generate theoretical tandem mass spectra of the predicted RiPP structures and compare them to experimental tandem MS data for RiPP discovery. The HypoRiPPAtlas was able to identify several cyclopeptide alkaloids including cyclopeptide alkaloid elaeagnin (Fig. 8) and lyciumins as a proof-of-concept.²¹ Orbitides can be characterized by tandem MS in a similar fashion by comparison of tandem mass spectra to predicted precursor peptides identified in plant transcriptomes or by de novo sequencing.³⁹⁸ While mass spectrometry provides new plant RiPP candidates in structure-guided discovery approaches, NMR or crystallography are usually applied on scaled peptide samples to determine new structures of candidate peptides.^88,90 With growing plant metabolomic databases, mass spectrometry-guided approaches will further drive plant RiPP discovery.

6.3. Gene-guided discovery

The characterization of precursor peptide genes (Table 1) and the growth of plant genetic resources^25,399–401 has enabled the discovery of plant RiPPs through bioinformatic prediction of peptide chemotypes from plant genes.

Cyclotides were discovered in Petunia plants by searching for cyclotide precursor peptides in the EST database of the NCBI.¹⁸² Many CRPs have been predicted in plant transcriptomes by bioinformatic analyses, for instance, identification of a new thionin from Papaver somniferum.^190,402 Similarly, genome mining and transcriptome analysis for fused burpitide precursor peptides has led to the discovery of new lyciumins, cyclopeptide alkaloids, and moroidins^17,19 and split burpitide precursor peptides that resulted in discovery of new cyclopeptide alkaloids and hibispeptins.²⁰ The characterization of a precursor peptide in any gene-guided discovery experiment yields a candidate core peptide sequence which can facilitate chemical characterization, e.g. by providing the long sequence information of cyclotides and CRPs. In addition, precursor peptides sequences can provide information about their processing through the secretory pathway via signal peptide prediction with SignalP.⁴⁰³ A challenge for gene-guided discovery of plant RiPPs is the identification of new precursor peptides and class-defining PTM enzymes in genomes with less biosynthetic gene clustering than in bacteria. A general approach to overcome this challenge is to search for core peptide sequences in predicted ORFs of peptide source plants^{4,5,7,12–14,17,52} or identify candidate precursor peptides based on genomic co-localization with scaffold-generating RiPP PTM genes.²⁰ Finally, tissue-specific paired transcriptomics and metabolomics has proven to be an essential approach for elucidating pathways for which biosynthetic enzymes are not co-localized.⁴⁰⁴

7. Future directions

Plant peptides offer exciting research questions in terms of their discovery and biosynthesis and opportunities for further biotechnological and medicinal applications. Like microbial RiPP classes, the field of plant RiPPs is experiencing a gold rush in peptide discovery fueled by increasing genetic resources such as the 1KP database,⁴⁰⁰ the 10KP project,²⁵ Phytozome⁴⁰¹ and metabolomic databases such as GNPS.³⁸⁴ While many RiPPs have been predicted based on bioinformatic studies, an important direction is the integration of omics-datasets for automated plant RiPP discovery as it has been organized for microbial natural products⁴⁰⁵ and chemical characterization of predicted RiPPs. In addition, discovery of plant RiPPs will depend on identification of new precursor peptides such as fused and split burpitide precursor peptides. Given the localization of some RiPP core peptides within larger plant proteins, this identification might require larger theoretical databases to match candidate peptide spectral data to core peptides within proteins encoded in a plant transcriptome or genome. Regarding sequencing data, it will be important to further improve de novo assembly for generation of repetitive precursor genes. Regarding mass spectrometry, tandem mass spectrometry experiments could further improve to generate more core peptide sequence data for de novo or gene-aided structure elucidation. Remaining biosynthetic questions of plant RiPPs include elucidating the origin of known plant-sourced peptides such as bouvardins and whether non-ribosomal peptide biosynthesis exists within the plant kingdom. In terms of biosynthesis, the several processing proteases of head-to-tail-cyclic peptides are still unknown. For burpitides, structural and mechanistic insight into macrocyclizations by burpitide cyclases is lacking and major biosynthetic questions regarding processing of both split and fused burpitide precursor are unanswered including reactions involved in proteolysis and terminal core-peptide modifications. With the combination of rapidly developing genomic and biosynthetic insights, the future of plant RiPPs holds great promise of the discovery of new bioactive molecules, enzyme reactions, and engineering platforms.

8. Conflicts of interest

The authors declare no conflicts of interest.

9. Acknowledgments

This work was partially supported by the US National Institutes of Health (R35GM147439 to J. R. C., F32GM146395 to L. S. M., R35GM146934 to R. D. K). We thank Sanath K. Kandy for generating a graphical abstract of this review.

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