Novel Cyclic Peptides in Seed of Annona muricata are Ribosomally

Synthesized

Mark F. Fisher,

†

Jingjing Zhang,

†

Oliver Berkowitz,

‡

James Whelan,

‡

and Joshua S. Mylne

†

The University of Western Australia,

School of Molecular Sciences & ARC Centre of Excellence in

Plant Energy Biology, 35 Stirling Highway, Crawley WA 6009, Australia

‡

Department of Animal, Plant and Soil Sciences, School of Life Sciences & ARC Industrial

Transformation Research Hub in Medicinal Agriculture, AgriBio building, La Trobe University,

Bundoora 3086, VIC, Australia

ABSTRACT: Small, cyclic peptides are reported to have many bioactivities. In bacteria and fungi they

can be made by non-ribosomal peptide synthetases, but in plants they are exclusively ribosomal.

Cyclic peptides from the Annona genus possess cytotoxic and anti-inflammatory activities, but their

biosynthesis is unknown. The medicinal soursop plant, Annona muricata, contains annomuricatins A

(cyclo-PGFVSA) and B (cyclo-PNAWLGT). Here, using de novo transcriptomics and tandem mass

spectrometry, we identify a suite of short transcripts for precursor proteins for ten validated

annomuricatins, nine of which are novel. In their precursors, annomuricatins are preceded by an

absolutely conserved Glu and each peptide sequence has a conserved proto-C-terminal Pro,

revealing parallels with the segetalin orbitides from the seed of Vaccaria hispanica, which are

processed through ligation by a prolyl oligopeptidase in a transpeptidation reaction.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

Plants express a number of different types of cyclic peptides. There are several families of homodetic

cyclic peptides (i.e. peptides cyclized by linking of their N- and C-termini) in plants whose

biosynthesis has been well-characterized. These are the cyclotides,

1, 2

cyclic knottins,

and PawS-

derived peptides (PDPs),

4, 5

all of which are stabilized by disulfide bridges and are cyclized by a

cysteine protease called asparaginyl endopeptidase (AEP).

4, 6-9

As such, these families of cyclic

peptides are known as ribosomally-synthesized and post-translationally modified peptides (RiPPs).

There is another group of homodetic cyclic peptides in plants, known as orbitides. These are also

RiPPs, but have no disulfide bonds and vary from five to 16 amino acid residues, although most are

7-9 residues.

10, 11

Orbitides are also considered to be RiPPs, but much less is known about their

biosynthesis than the peptide families mentioned above. One family of orbitides, the PawL-derived

peptides (PLPs), are closely related to the PDPs, and probably cyclized by the same mechanism,

12, 13

but most other orbitides do not contain Asp or Asn residues, which are required for cyclisation by

AEP.

Thus most orbitides probably have different mechanisms of cyclisation to PLPs.

The genes encoding orbitides are known only in a small number of cases. Condie et al.

demonstrated that the segetalins, a group of orbitides found in the seeds of Vaccaria hispanica

(Saponaria vaccaria L., Caryophyllaceae), are individually encoded by short genes that express

propeptides 30-40 amino acids long. The researchers also showed evidence of a genetic origin for

orbitides in some Citrus species. Okinyo-Owiti et al.

characterized three novel orbitides from flax

(Linum usitatissimum L., Linaceae), called cyclolinopeptides 17-19, and found that these

cyclolinopeptides (more recently called linusorbs

) are derived from gene-encoded precursors, in

which multiple peptides are encoded in a single gene. A search of expressed sequence tags from

Jatropha curcas (Euphorbiaceae) suggested that curcacyclines A and B are genetically encoded.

these species the cyclic peptide is excised from a highly conserved N-terminal leader sequence and

C-terminal follower sequence (though the Jatropha peptides appear to have no follower sequence).

The core peptides (the linear precursors of the final cyclic peptide) show little conservation, except

at their N- and C-termini.

There are many other orbitides whose biosyntheses are not known. These include the ~35 orbitides

found in plants of the Annonaceae family, including some reported to possess cytotoxic

17, 18

and anti-

inflammatory

19, 20

activities. Two of these orbitides are found in the seeds of Annona muricata.

Annomuricatin A (cyclo-GPFVSA; monoisotopic mass 558.28 Da) was characterized by Li et al.

and

annomuricatin B (cyclo-PNAWLGT) by the same group.

A third orbitide, annomuricatin C, was

reported by Wélé et al.

but was later shown by structural studies to be identical to annomuricatin

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

We were interested in the genetic origins of the annomuricatins, which we investigated by

combining de novo transcriptomics with peptide tandem mass spectrometry. Having sequenced a

novel orbitide, annomuricatin D, from tandem mass spectrometry data, we were able to identify a

single transcript encoding both annomuricatin D and the previously-known annomuricatin A, thus

demonstrating that annomuricatins are ribosomally synthesized. We were then able to identify

transcripts encoding a further eight novel annomuricatins (E-L) and sequenced them by mass

spectrometry.

RESULTS AND DISCUSSION

To characterize the biosynthesis of the annomuricatins, total RNA was extracted from A. muricata

seeds, next generation RNA sequencing (RNA-seq) performed and a transcriptome assembled using

established methods.

Using cyclic permutations of the two known peptides annomuricatin A and

annomuricatin B, the transcriptome was searched for sequences encoding them; there were

hundreds of contigs that had the potential to encode annomuricatin A, but we found none that

could encode annomuricatin B.

Figure 1. Total ion current chromatogram for extracts of A. muricata seeds with the m/z values

and the acquisition times of the largest peaks labelled. All ions shown are [M+H]

A liquid chromatography-mass spectrometry (LC-MS) analysis of seed peptide masses for A. muricata

revealed a mass consistent with the presence of annomuricatin A, no mass for annomuricatin B, but

critically it revealed several other seemingly abundant masses (Figure 1). One of these, eluting at

24.7 min, was especially abundant and the protonated molecule had a measured m/z of 946.515

[M+H]

(Figure 2A). Treatment of the sample with 1.2 M hydrochloric acid followed by liquid

chromatography-tandem mass spectrometry (LC-MS/MS)

caused the formation of a new

compound with a molecular weight ~963 Da, as shown by peaks at m/z 482.767 [M+2H]

and

964.524 [M+H]

, although the original peak (now measured at m/z 946.514) remained (Figure 2B). If

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

the 946.5 m/z ion was a novel annomuricatin, this is consistent with partial acid hydrolysis of its

peptide backbone leading to an 18 Da mass increase.

Figure 2. Combined extracted ion chromatograms at m/z 946.515 and m/z 964.526 for peptide

extracts of A. muricata seeds. (A) Native extract; (B) After treatment with dilute hydrochloric

acid. Insets show the mass spectrum at each peak in the chromatogram.

Peptide evidence for annomuricatin D

Working on the hypothesis that the m/z 946.5 ion was a novel annomuricatin, we sequenced the

protonated molecule of the hydrolyzed product (m/z 964.5) from MS/MS data to give the sequence

SXFPPXPGH (where X represents either of the two isobaric residues Leu or Ile) (Figure 3),

corresponding to an exact m/z of 964.526 for the acyclic peptide. The order of the Ser and Ile/Leu

residues at the N-terminus could not be determined unambiguously by MS/MS, however. We

tentatively named this novel cyclic peptide annomuricatin D.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

Figure 3. MS/MS confirmation of annomuricatin D. Partial hydrolysis using hydrochloric acid

gave a sequence of b and y ions, with the ring breakage C-terminal to the His residue (inset

shows the peptide sequence). Note that the order of the Ser and Ile/Leu residues could not be

unambiguously determined.

Identification of novel transcripts encoding annomuricatins

Returning to search the A. muricata transcriptome using tBLASTn with all 72 possible annomuricatin

D query sequences (i.e. all circular permutations of the two possible 9-residue sequences with four

possible Ile/Leu combinations) produced just one contig with a perfect match, namely GHSIFPPIP. In

the contig matching annomuricatin D, we observed that the complete ORF also encoded

annomuricatin A (Figure 4).

Figure 4. Searches of the A. muricata transcriptome by tBLASTn using annomuricatin D as the

query sequence revealed a contig whose ORF encoded annomuricatin D as well as the

previously published annomuricatin A.

Within this predicted precursor protein, the annomuricatin sequences both ended with Pro and

were both preceded by Glu, consistent with the hypothesis that their proteolytic maturation was

conserved and involved two different enzymes – one targeting Glu that would release the N-

terminus of the core peptide and a protease that targeted Pro that would probably perform a

cleavage-coupled transpeptidation to the freed N-terminus to form a cyclic annomuricatin in a

manner similar to other cyclic RiPPs. The sequence for annomuricatin A was N-terminal to

annomuricatin D in the encoded sequence and so we named the transcript ProannomuricatinAD,

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

abbreviated to PamAD. Using the PamAD transcript as a tBLASTn query, five similar coding

sequences with the potential to encode nine annomuricatins were discovered, with a maximum

expected value of 4x10

-3

Figure 5. Manual alignment of the translated sequences of Proannomuricatin genes from the

transcriptome of A. muricata seeds. The putative annomuricatin core peptide sequences are

separated from the leader and follower sequences with spaces and have a green background.

The absolutely conserved Glu in the P1 position is shown with a yellow background. Note the

absolutely conserved Pro (pink background) which may be significant in processing of the

peptide precursors, after cleavage at the N-terminus of each core peptide.

Consistent with the lack of peptide mass evidence, no evidence was found for a transcript matching

the previously reported annomuricatin B sequence.

This variation in peptide content is something

that may be due to environmental differences in transcript expression.

26, 27

To confirm the sequences

obtained by RNA-seq and assembly of contigs, primers were designed against the end of each contig

to amplify a full length ORF and used genomic DNA as the template. We were able to amplify all six

genes and found them to be intronless and a 100% match to the contigs assembled by RNA-seq.

Confirmation of annomuricatins E to L by tandem mass spectrometry

Knowing the sequence of potential peptides facilitates their confirmation by LC-MS/MS, even

without having to linearize the peptides before MS/MS analysis. Using the gene-encoded sequences,

we predicted the expected mass for each peptide and could identify masses that matched

predictions in all the putative Pam genes (Table 1). These putative masses named annomuricatin E to

L were each sequenced by LC-MS/MS (Figures S1-S10). Annomuricatins G and I were found in two

forms having either a reduced (Figures S3 and S6 respectively) or oxidized methionine (Figures S4

and S7). It is not possible to say whether this is a biologically relevant post-translational modification

or occurred during peptide extraction and purification. We have therefore not classified the two

forms as separate peptides.

Although peaks were found in the mass spectrum corresponding to a second putative annomuricatin

in the PamL transcript, MS/MS data were not consistent with the predicted sequence from the

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

transcript (Figure S11). We also searched for a possible second peptide in the PamG transcript, but

none of the sequences searched was found in the mass spectrometry data.

Table 1. Summary of annomuricatin cyclic peptides, the genes that encode each and

evidence for the peptides from mass spectrometry. N.D. = not detected.

Annomuricatin

Sequence

Gene

Peptide evidence

A (also C)

GFVSAP

PamAD

Li et al.

NAWLGTP

N.D.

GHSIFPPIP

PamAD

Figure 2, Figure 3

GLVTVP

PamEF

Figure S1

GLSAVTP

PamEF

Figure S2

MIGDYSYP

PamG

Figure S3, Figure S4

TLVPGYSP

PamHI

Figure S5

GFMHSPVP

PamHI

Figure S6, Figure S7

GPPLYLA

PamJK

Figure S8

RPFDFLFP

PamJK

Figure S9

GLGIYP

PamL

Figure S10

Annomuricatin B, which could not be detected at either the peptide or transcript level, was

proposed by Li et al.

to have the sequence cyclo-PNAWLGT. Based on the conservation of

Pro at the C-terminus of the core peptide we have written the sequence as NAWLGTP.

Annomuricatin G (cyclo-MIGDYSYP) was found in two forms – one with standard Met

(Figure S3) and the other with an oxidized-Met (Figure S4).

Annomuricatin I (cyclo-GFMHSPVP) was found in two forms – one with standard Met

(Figure S6) and the other with an oxidized-Met (Figure S7).

Like many other cyclic peptides, most of the core peptide sequence is not conserved, except for the

N-terminal and C-terminal residues. In the case of the annomuricatins, the N-terminus is most often

Gly, which tends to be the case in the majority of cyclic RiPPs, and the C-terminus residue is Pro or,

in one case, Ala, both of which can be cleaved by a prolyl oligopeptidase.

In the leader sequence,

Glu in the P1 position to the core peptide is absolutely conserved and the P2 residue varies, but is

most often Ser. Assuming that the precursor peptide is cleaved at the proto-N-terminus of each of

the two core peptides prior to cyclization, as is thought to be the case for the segetalins of Vaccaria

hispanica,

then the only other very highly conserved residue is found at the fourth residue from

the C-termini of the two propeptides thus formed. This residue is invariably Pro (Figure 5), and it is

tempting to speculate, despite its distance from the core peptide, that this is in some way required

for the prolyl oligopeptidase to cyclize the peptide.

The annomuricatins described here are typical in size for orbitides at between six and nine residues.

Many annomuricatins contain mainly hydrophobic residues such as Val, Gly, Ala, Leu and Ile, which

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

again is typical of orbitides.

Annomuricatin K is unusual in that it has an Arg and an Asp residue.

Both residues are rare in most orbitides except for the PLPs, which have an absolutely conserved Asp

at the C-terminus of the core peptide, essential for their macrocyclization.

Other annomuricatin-like peptides

The number of similar annonomuricatins suggests the genes encoding them have duplicated and

diverged. To investigate whether this type of gene is more widespread among the Annonaceae,

RNA-seq data were downloaded from the NCBI Sequence Read Archive. These data were generated

from RNA isolated from the mature flowers of Annona squamosa

and leaves of A. muricata.

assembled transcriptomes and searched them with tBLASTn using the pamAD transcript as the query

sequence. This approach identified several contigs with strong sequence similarity to Pam genes.

Three candidate transcripts were identified in A. muricata leaves with a maximum expected value of

1.6x10

-3

, which were different from those in the A. muricata seeds, encoding a total of six putative

peptides (Figure 6A). Ten candidate transcripts from A. squamosa (maximum expected value 2.7x10

) were also found, most encoding two possible peptides, though several varied considerably from

the canonical sequence, with the putative core peptide lacking a C-terminal Pro or Ala (Figure 6B).

Figure 6. Alignments of transcripts from (A) A. muricata leaves and (B) A. squamosa flowers.

Putative core peptides are shown with a green background, the absolutely conserved Glu

with a yellow background, and the highly conserved Pro or Ala in pink. Asterisks are stop

codons, shown where the ORF is complete. The last sequence shown corresponds to the

known cyclic peptide, cyclosquamosin D,

and appears to be only a fragment of the full ORF.

The cases where the putative core peptides do not have Pro or Ala at the C-terminus may represent

genes where one of the two peptides encoded has degenerated into a non-functional sequence, as

appears to have occurred in the PamG and PamL transcripts from A. muricata seeds. Again, the Glu

in the P1 position at the N-terminus of the core peptide is absolutely conserved. The highly

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

conserved Pro in the follower sequence is again prominent, though sometimes replaced by Ala. In

one case it was in the fifth position from the C-terminus rather than the fourth. Without a tissue

sample on which to perform MS/MS analysis, it was impossible to say whether these differences

from the A. muricata seed sequences prevent production of the cyclic peptide.

What can be seen in these homologs of the Pam genes of A. muricata seeds is that similar transcripts

are present in more than one Annonaceae species. The putative propeptides encoded by the

transcripts have a high degree of sequence similarity and most of them appear to encode two cyclic

peptides. We therefore suggest this type of orbitide-encoding gene could occur across the

Annonaceae and would be an interesting topic for further research. It is also noteworthy that the

putative peptides from the leaves of A. muricata are quite different to the seed peptides, suggesting

the annomuricatins could have organ-specific functions.

Biosynthesis of annomuricatins

The Proannomuricatin transcripts are short (~200 nt) and the leader and follower sequences around

the core peptide are highly conserved (Figure 5). The core peptide sequence is highly variable, but

the N-terminus has a highly conserved Gly, preceded by an absolutely conserved Glu in the P1

position. The C-terminal Pro of the core peptide is also absolutely conserved, except for the

presence of Ala in one instance.

Much work has been done on the biosynthesis of plant cyclic peptides that rely on AEP for their

maturation and cyclisation.

4-8, 33

A similar depth of understanding exists for the orbitide segetalin A

and its relatives, whose cyclization of a conserved C-terminal Ala residue is performed by the prolyl

oligopeptidase PCY1.

14, 29

Prior to cyclization, the segetalins are cleaved at the N-terminus of the core

peptide by an as-yet-uncharacterized enzyme, OLP1,

which presumably is able to recognize the

conserved Gln or Glu residue at the P1 position. Another example of peptides cyclized by a prolyl

oligopeptidase are the amanitins from the fungus Galerina marginata; these are cyclized by

GmPOPB. Unlike the segetalins, pro-amanitin is cleaved by POPB at the N-terminus of the core

peptide as well as at the C-terminus due to the conserved Pro at the P1 position to the N-terminus.

Based on the conserved residues in Pam genes and parallels with other cyclic peptide biosyntheses,

annomuricatin precursors are likely to be cleaved first at the core peptide N-terminus by a Glu-

targeting protease and then cleaved at the C-terminus and ligated by a POP. This may parallel the

action of OLP1 and PCY1 in V. hispanica, since the former appears to target Glu or Gln at the proto-

N-terminus, and the latter cleaves at Ala or Pro.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

Here we have shown that one known and nine novel annomuricatins in the seeds of A. muricata are

encoded by six very similar short genes; four of them encode two cyclic peptides, and the other two

encode one peptide each. Similar genes are also found in the leaves of A. muricata and flowers of A.

squamosa, indicating that such cyclic peptides may be present in other Annonaceae species.

Comparison with the segetalins of V. hispanica indicates that a prolyl oligopeptidase is the likely

cyclisation agent. Further study is required to identify this POP and to characterize its structure and

mechanism of action.

EXPERIMENTAL SECTION

Plant material

Seeds of A. muricata were purchased from B & T World Seeds (Paguignan, France). While under

quarantine, seeds were treated with Gaucho 600 insecticide (Bayer CropScience) to comply with

Western Australian regulations. Soon after, seeds were frozen in liquid nitrogen, ground to a fine

powder and stored at -80 °C until required.

Seed peptide extraction

Seed peptides were extracted as previously described.

Briefly, peptides were extracted in 50%

MeOH / 50% CHCl

. Phases were separated by the alternate addition of CHCl

and 0.05%

trifluoroacetic acid. The upper, aqueous phase was dried overnight in a vacuum centrifuge

(Labconco) prior to purification.

Purification of seed peptide extracts

Seed peptide extracts were purified according to the method previously described.

Briefly, the

crude extract was purified by solid-phase extraction using a 30 mg Strata-X polymeric reversed-

phase column (Phenomenex). The extract was applied to the column as an aqueous solution of 5%

MeCN (v/v) / 0.1% formic acid (v/v), then purified peptides were eluted with 85% MeCN (v/v) / 0.1%

formic acid (v/v). The extract was dried in a vacuum centrifuge and redissolved in 5% MeCN (v/v) /

0.1% formic acid (v/v) for LC-MS analysis. HPLC-grade solvents were used throughout (Honeywell).

LC-MS/MS for peptide sequencing

Samples (2 µL) were injected onto an EASY-Spray PepMap C18 column (75 μm x 150 mm, 3 μm

particle size, 10 nm pores; Thermo Fisher Scientific) using a Dionex UltiMate 3000 nano UHPLC

system (Thermo Fisher Scientific) at flow rate of 200 nL/min by the “µL pick-up” method. A gradient

elution was run from 5% solvent B to 95% solvent B over 40 minutes. Solvent A was 0.1% formic acid

in water and solvent B was 0.1% formic acid in MeCN (Fisher Scientific). The resulting electrospray

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

(source voltage 1,800 V) was analyzed by an Orbitrap Fusion mass spectrometer (Thermo Fisher

Scientific) running in positive ionization, data-dependent, “top speed” MS/MS mode, employing the

Orbitrap mass analyzer for both MS and MS/MS measurements at a resolution of 120,000 for MS

and 60,000 for MS/MS. Parameters were set as follows: HCD fragmentation alternating between

14% ± 3% and 23% ± 3% energy, MS scan range from 400 to 1600 m/z, minimum MS/MS m/z 50,

isolation window 1.2, ACG 400,000 (MS) and 500,000 (MS/MS), maximum injection time 200 ms

(MS) and 250 ms (MS/MS) and 2 microscans for MS/MS. Only ions with an intensity > 100,000 were

fragmented for MS/MS.

Peptide sequencing

Peptides were sequenced by visual examination of MS/MS spectra, aided by fragment predictions

from the program mMass

for cyclic peptides and MS Product on the ProteinProspector website for

acyclic peptides.

The parameters used for MS-Product selected a, b, y and immonium ions, plus

internal fragments. Neutral losses were set to water (when S, T, E or D present) or ammonia (when

R, K, Q or N present). Other parameters were left at their default values. Similar options were chosen

for mMass except that y ions were not selected; these do not appear in the mass spectra of cyclic

peptides because such peptides lack a carboxyl-terminus.

Annona muricata RNA-seq and transcriptome assembly

Seeds of A. muricata (100 mg) were ground to a powder using a mortar and pestle cooled with liquid

nitrogen. Total RNA was isolated using the Spectrum Plant Total RNA kit (Sigma Aldrich) and quality

validated on a TapeStation 2200 system (Agilent). RNA-seq libraries were generated using the

TruSeq Stranded Total RNA with Ribo-Zero Plant kit according to the manufacturer’s instructions

(Illumina) and sequenced on a NextSeq 550 system (Illumina) as paired-end reads with a length

of150 bp and an average quality score (Q30) of above 90%. The raw reads were deposited in the

NCBI Sequence Read Archive under accession number SRR8959862.

The A. muricata transcriptome was assembled using CLC Genomics Workbench 10.0.1 (QIAGEN

Aarhus A/S). The raw reads were trimmed to a quality threshold of Q30 and minimum length 50, and

the assembly was performed with word size 64 and minimum contig length 200. Other parameters

remained at their default values.

Other Annona transcriptome assemblies

RNA-seq paired-read data were downloaded from the Sequence Read Archive of the National

Institutes of Health for A. squamosa mature flowers (run SRR3478571) and A. muricata leaves (run

ERR2040135). Using CLC Genomics Workbench 11.0, transcriptomes were assembled for each of

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

these datasets. For A. squamosa, the raw paired-end reads were trimmed to a quality threshold of

22 and minimum length 50, and the assembly was performed with word size 64, minimum contig

length 50 and bubble size 100. For A. muricata leaves, the raw paired-end reads were trimmed to a

quality threshold of 20 and minimum length 50, and the assembly was performed with word size 50

and minimum contig length 50. In both cases, all other parameters remained at their default values.

BLAST searches of transcriptomic data

A BLAST database was constructed from each of the assembled transcriptomes using CLC Genomic

Workbench. To search for proannomuricatin peptide sequences, the A. muricata database was

searched using tBLASTn with the PamAD peptide sequence as a query. The substitution matrix used

was PAM45 to find only closely related sequences. Other parameters selected were: maximum

expected value 1, gap open cost 15, gap extend cost 2 and word size 3. Similar searches were carried

out on transcriptomes of A. muricata leaves and A. squamosa flowers downloaded from the SRA.

Cloning of Proannomuricatin genes

Genomic DNA was extracted from 2 g of frozen A. muricata seed powder with the DNEasy Mericon

Food Kit (QIAGEN) according to the manufacturer’s instructions. DNA was quantified using a

NanoDrop 2000 (Thermo Fisher Scientific).

The genomic DNA template was amplified by the polymerase chain reaction (PCR) using Pfu Ultra

High-Fidelity DNA polymerase (Agilent Technologies). Each 50 μL reaction consisted of genomic DNA

(~12 ng), 5 μL Pfu Ultra DNA polymerase reaction buffer (10x), 400 μM mixed dNTPs, 0.5 μL Pfu Ultra

DNA polymerase and 0.4 μM of each of the appropriate forward and reverse primers (Table S1).PCR

amplification was performed in a Veriti 96-well thermocycler (Applied Biosystems) programmed as

follows: 95 °C for 2 min followed by 5 cycles of 95 °C for 30 s; 65 °C for 30 s; 72 °C for 30 s then 30

cycles of 95 °C for 30 s; 60 °C for 30 s; 72 °C for 30 s; and finally 72 °C for 10 min (PamAD, PamEF,

PamG), or 95 °C for 2 min followed by 35 cycles of 95 °C for 30 s; 60 °C for 30 s; 72 °C for 30 s; and

finally 72 °C for 10 min (PamHI, PamJK, PamLM).

PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) according to the

manufacturer’s instructions. DNA was eluted in 30 µL of water, quantified on a NanoDrop 2000 and

sent for dideoxy sequencing using the forward PCR primers mentioned above (Garvan Institute,

Darlinghurst NSW, Australia).

The six Proannomuricatin (Pam) genes from this study were deposited in GenBank under accession

numbers MK836460-MK836465.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

AUTHOR INFORMATION

Corresponding author

Joshua S. Mylne, School of Molecular Sciences & ARC Centre of Excellence in Plant Energy Biology,

The University of Western Australia, 35 Stirling Highway, Crawley, Perth 6009, Australia. E-mail:

joshua.mylne@uwa.edu.au

Author Contributions

M.F.F. and J.S.M. conceived the study; O.B. and J.W. performed RNA-seq; J.Z. and M.F.F. assembled

the A. muricata transcriptome; M.F.F performed all other experiments and analyzed data; M.F.F and

J.S.M. wrote the manuscript with help from all other authors.

Funding Sources

M.F.F. was supported by the Australian Research Training Program and a Bruce and Betty Green

Postgraduate Research Scholarship. J.Z. was supported by an International Postgraduate Research

Scholarship and a University Postgraduate Award from The University of Western Australia. J.S.M.

was supported in part by an ARC Future Fellowship (FT120100013). This work was supported by ARC

grant DP190102058 to J.S.M. and CE140100008 to J.W.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

The authors thank Nicolas L. Taylor of the School of Molecular Sciences at the University of Western

Australia for providing advice and assistance in mass spectrometry aspects of this project.

The authors acknowledge the facilities, and the scientific and technical assistance of the Australian

Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation &

Analysis, The University of Western Australia, a facility funded by the University, State and

Commonwealth governments.

REFERENCES

(1) Saether, O.; Craik, D. J.; Campbell, I. D.; Sletten, K.; Juul, J.; Norman, D. G. Biochemistry 1995, 34,

4147-4158.

(2) Craik, D. J.; Daly, N. L.; Bond, T.; Waine, C. J. Mol. Biol. 1999, 294, 1327-1336.

(3) Hernandez, J. F.; Gagnon, J.; Chiche, L.; Nguyen, T. M.; Andrieu, J. P.; Heitz, A.; Trinh Hong, T.;

Pham, T. T.; Le Nguyen, D. Biochemistry 2000, 39, 5722-5730.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

(4) Mylne, J. S.; Colgrave, M. L.; Daly, N. L.; Chanson, A. H.; Elliott, A. G.; McCallum, E. J.; Jones, A.;

Craik, D. J. Nat. Chem. Biol. 2011, 7, 257-259.

(5) Elliott, A. G.; Delay, C.; Liu, H.; Phua, Z.; Rosengren, K. J.; Benfield, A. H.; Panero, J. L.; Colgrave, M.

L.; Jayasena, A. S.; Dunse, K. M.; Anderson, M. A.; Schilling, E. E.; Ortiz-Barrientos, D.; Craik, D. J.;

Mylne, J. S. Plant Cell 2014, 26, 981-995.

(6) Gillon, A. D.; Saska, I.; Jennings, C. V.; Guarino, R. F.; Craik, D. J.; Anderson, M. A. Plant J. 2008, 53,

505-515.

(7) Mylne, J. S.; Chan, L. Y.; Chanson, A. H.; Daly, N. L.; Schaefer, H.; Bailey, T. L.; Nguyencong, P.;

Cascales, L.; Craik, D. J. Plant Cell 2012, 24, 2765-2778.

(8) Bernath-Levin, K.; Nelson, C.; Elliott, A. G.; Jayasena, A. S.; Millar, A. H.; Craik, D. J.; Mylne, J. S.

Chem. Biol. 2015, 22, 571-582.

(9) Saska, I.; Gillon, A. D.; Hatsugai, N.; Dietzgen, R. G.; Hara-Nishimura, I.; Anderson, M. A.; Craik, D.

J. J. Biol. Chem. 2007, 282, 29721-29728.

(10) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni, T. S.; Bulaj, G.; Camarero, J. A.;

Campopiano, D. J.; Challis, G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.;

Donadio, S.; Dorrestein, P. C.; Entian, K.-D.; Fischbach, M. A.; Garavelli, J. S.; Goransson, U.;

Gruber, C. W.; Haft, D. H.; Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars, M.;

Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.; Marahiel, M. A.; Mitchell, D. A.; Moll,

G. N.; Moore, B. S.; Muller, R.; Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.;

Patchett, M. L.; Piel, J.; Reaney, M. J. T.; Rebuffat, S.; Ross, R. P.; Sahl, H.-G.; Schmidt, E. W.;

Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.;

Tang, G.-L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S. C.; Willey, J. M.;

van der Donk, W. A. Nat. Prod. Rep. 2013, 30, 108-160.

(11) Fisher, M. F.; Payne, C. D.; Rosengren, K. J.; Mylne, J. S. J. Nat. Prod. 2019, 82, 2152-2158.

(12) Jayasena, A. S.; Fisher, M. F.; Panero, J. L.; Secco, D.; Bernath-Levin, K.; Berkowitz, O.; Taylor, N.

L.; Schilling, E. E.; Whelan, J.; Mylne, J. S. Mol. Biol. Evol. 2017, 34, 1505-1516.

(13) Fisher, M. F.; Zhang, J.; Taylor, N. L.; Howard, M. J.; Berkowitz, O.; Debowski, A. W.; Behsaz, B.;

Whelan, J.; Pevzner, P. A.; Mylne, J. S. Plant Direct 2018, 2, e00042.

(14) Condie, J. A.; Nowak, G.; Reed, D. W.; John Balsevich, J.; Reaney, M. J. T.; Arnison, P. G.; Covello,

P. S. Plant J. 2011, 67, 682–690.

(15) Okinyo-Owiti, D. P.; Young, L.; Burnett, P.-G. G.; Reaney, M. J. T. Peptide Science 2014, 102, 168-

175.

(16) Shim, Y. Y.; Young, L. W.; Arnison, P. G.; Gilding, E.; Reaney, M. J. T. J. Nat. Prod. 2015, 78, 645-

652.

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

(17) Wélé, A.; Landon, C.; Labbe, H.; Vovelle, F.; Zhang, Y.; Bodo, B. Tetrahedron 2004, 60, 405-414.

(18) Wélé, A.; Zhang, Y.; Dubost, L.; Pousset, J.-L.; Bodo, B. Chem. Pharm. Bull. (Tokyo) 2006, 54, 690-

692.

(19) Chuang, P. H.; Hsieh, P. W.; Yang, Y. L.; Hua, K. F.; Chang, F. R.; Shiea, J.; Wu, S. H.; Wu, Y. C. J.

Nat. Prod. 2008, 71, 1365-70.

(20) Yang, Y. L.; Hua, K. F.; Chuang, P. H.; Wu, S. H.; Wu, K. Y.; Chang, F. R.; Wu, Y. C. J. Agric. Food

Chem. 2008, 56, 386-92.

(21) Li, C.; Tan, N.; Lu, Y.; Liang, H.; Mu, Q.; Zheng, H.; Hao, X.; Zhou, J. Acta Botanica Yunnanica

1995, 17, 459-462.

(22) Li, C.; Tan, N.; Zheng, H.; Mu, Q.; Hao, X.; He, Y.; Zou, J. Phytochemistry 1998, 48, 555-556.

(23) Wélé, A.; Zhang, Y.; Caux, C.; Brouard, J.-P.; Pousset, J.-L.; Bodo, B. C. R. Chim. 2004, 7, 981-988.

(24) Wu, L.; Lu, Y.; Zheng, Q.-T.; Tan, N.-H.; Li, C.-M.; Zhou, J. J. Mol. Struct. 2007, 827, 145-148.

(25) Fisher, M. F.; Mylne, J. S. J. Proteome Res. 2019, 18, 4065-4071.

(26) Gui, B.; Shim, Y. Y.; Datla, R. S. S.; Covello, P. S.; Stone, S. L.; Reaney, M. J. T. J. Agric. Food Chem.

2012, 60, 8571-8579.

(27) Narayani, M.; Chadha, A.; Srivastava, S. J. Nat. Prod. 2017.

(28) Moriyama, A.; Nakanishi, M.; Sasaki, M. J. Biochem. 1988, 104, 112-117.

(29) Barber, C. J. S.; Pujara, P. T.; Reed, D. W.; Chiwocha, S.; Zhang, H.; Covello, P. S. J. Biol. Chem.

2013, 288, 12500-12510.

(30) Liu, K.; Feng, S.; Pan, Y.; Zhong, J.; Chen, Y.; Yuan, C.; Li, H. Front. Plant Sci. 2016, 7, 1695.

(31) Leebens-Mack, J. H.; Barker, M. S.; Carpenter, E. J.; Deyholos, M. K.; Gitzendanner, M. A.;

Graham, S. W.; Grosse, I.; Li, Z.; Melkonian, M.; Mirarab, S.; Porsch, M.; Quint, M.; Rensing, S. A.;

Soltis, D. E.; Soltis, P. S.; Stevenson, D. W.; Ullrich, K. K.; Wickett, N. J.; DeGironimo, L.; Edger, P.

P.; Jordon-Thaden, I. E.; Joya, S.; Liu, T.; Melkonian, B.; Miles, N. W.; Pokorny, L.; Quigley, C.;

Thomas, P.; Villarreal, J. C.; Augustin, M. M.; Barrett, M. D.; Baucom, R. S.; Beerling, D. J.;

Benstein, R. M.; Biffin, E.; Brockington, S. F.; Burge, D. O.; Burris, J. N.; Burris, K. P.; Burtet-

Sarramegna, V.; Caicedo, A. L.; Cannon, S. B.; Çebi, Z.; Chang, Y.; Chater, C.; Cheeseman, J. M.;

Chen, T.; Clarke, N. D.; Clayton, H.; Covshoff, S.; Crandall-Stotler, B. J.; Cross, H.; dePamphilis, C.

W.; Der, J. P.; Determann, R.; Dickson, R. C.; Di Stilio, V. S.; Ellis, S.; Fast, E.; Feja, N.; Field, K. J.;

Filatov, D. A.; Finnegan, P. M.; Floyd, S. K.; Fogliani, B.; García, N.; Gâteblé, G.; Godden, G. T.; Goh,

F.; Greiner, S.; Harkess, A.; Heaney, J. M.; Helliwell, K. E.; Heyduk, K.; Hibberd, J. M.; Hodel, R. G.

J.; Hollingsworth, P. M.; Johnson, M. T. J.; Jost, R.; Joyce, B.; Kapralov, M. V.; Kazamia, E.; Kellogg,

E. A.; Koch, M. A.; Von Konrat, M.; Könyves, K.; Kutchan, T. M.; Lam, V.; Larsson, A.; Leitch, A. R.;

Lentz, R.; Li, F.-W.; Lowe, A. J.; Ludwig, M.; Manos, P. S.; Mavrodiev, E.; McCormick, M. K.;

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint

McKain, M.; McLellan, T.; McNeal, J. R.; Miller, R. E.; Nelson, M. N.; Peng, Y.; Ralph, P.; Real, D.;

Riggins, C. W.; Ruhsam, M.; Sage, R. F.; Sakai, A. K.; Scascitella, M.; Schilling, E. E.; Schlösser, E.-

M.; Sederoff, H.; Servick, S.; Sessa, E. B.; Shaw, A. J.; Shaw, S. W.; Sigel, E. M.; Skema, C.; Smith, A.

G.; Smithson, A.; Stewart, C. N.; Stinchcombe, J. R.; Szövényi, P.; Tate, J. A.; Tiebel, H.; Trapnell,

D.; Villegente, M.; Wang, C.-N.; Weller, S. G.; Wenzel, M.; Weststrand, S.; Westwood, J. H.;

Whigham, D. F.; Wu, S.; Wulff, A. S.; Yang, Y.; Zhu, D.; Zhuang, C.; Zuidof, J.; Chase, M. W.; Pires, J.

C.; Rothfels, C. J.; Yu, J.; Chen, C.; Chen, L.; Cheng, S.; Li, J.; Li, R.; Li, X.; Lu, H.; Ou, Y.; Sun, X.; Tan,

X.; Tang, J.; Tian, Z.; Wang, F.; Wang, J.; Wei, X.; Xu, X.; Yan, Z.; Yang, F.; Zhong, X.; Zhou, F.; Zhu,

Y.; Zhang, Y.; Ayyampalayam, S.; Barkman, T. J.; Nguyen, N.-p.; Matasci, N.; Nelson, D. R.; Sayyari,

E.; Wafula, E. K.; Walls, R. L.; Warnow, T.; An, H.; Arrigo, N.; Baniaga, A. E.; Galuska, S.; Jorgensen,

S. A.; Kidder, T. I.; Kong, H.; Lu-Irving, P.; Marx, H. E.; Qi, X.; Reardon, C. R.; Sutherland, B. L.; Tiley,

G. P.; Welles, S. R.; Yu, R.; Zhan, S.; Gramzow, L.; Theißen, G.; Wong, G. K.-S.; One Thousand Plant

Transcriptomes, I. Nature 2019, 574, 679-685.

(32) Morita, H.; Sato, Y.; Kobayashi, J. i. Tetrahedron 1999, 55, 7509-7518.

(33) Saska, I.; Craik, D. J. Trends Biochem. Sci. 2008, 33, 363-368.

(34) Luo, H.; Hong, S.-Y.; Sgambelluri, R. M.; Angelos, E.; Li, X.; Walton, Jonathan D. Chem. Biol. 2014,

21, 1610-1617.

(35) Niedermeyer, T. H. J.; Strohalm, M. PLoS One 2012, 7, e44913.

(36) Baker, P. R.; Clauser, K. R. ProteinProspector. http://prospector.ucsf.edu/ (5th December 2019)

.CC-BY-NC-ND 4.0 International licenseavailable under a

was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted February 16, 2020. ; https://doi.org/10.1101/647552doi: bioRxiv preprint