Precursor processing for plant peptide hormone maturation by subtilisin-like serine proteinases

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Science  23 Dec 2016:
Vol. 354, Issue 6319, pp. 1594-1597
DOI: 10.1126/science.aai8550

Prohormone processing by subtilases

A flower that has gone to seed will drop its petals in a regulated process called abscission. Schardon et al. analyzed the production of the peptide hormone that regulates floral organ abscission in the model plant Arabidopsis thaliana. They used tissue-specific expression of proteinase inhibitors to identify the subtilisin-like proteinases that act as prohormone convertases required for peptide hormone production in plants.

Science, this issue p. 1594


Peptide hormones that regulate plant growth and development are derived from larger precursor proteins by proteolytic processing. Our study addressed the role of subtilisin-like proteinases (SBTs) in this process. Using tissue-specific expression of proteinase inhibitors as a tool to overcome functional redundancy, we found that SBT activity was required for the maturation of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION), a peptide signal for the abscission of floral organs in Arabidopsis. We identified three SBTs that process the IDA precursor in vitro, and this processing was shown to be required for the formation of mIDA (the mature and bioactive form of IDA) as the endogenous signaling peptide in vivo. Hence, SBTs act as prohormone convertases in plants, and several functionally redundant SBTs contribute to signal biogenesis.

Small posttranslationally modified peptides function as extracellular signaling molecules in plants (1). Such peptides control plant growth and development, as well as interactions between plants and their environment (1, 2). Addressing the biogenesis of plant peptide hormones, we focused on a peptide that serves as a signal for the abscission of floral organs (petals, sepals, and stamens).

Abscission is on full display during autumn, when deciduous trees shed their leaves. The abscission process is also agriculturally important, as it facilitates the dispersal of fruits and seeds. In most flowering plants, including Arabidopsis thaliana, floral organs abscise when pollination is complete (3, 4). The IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) gene controls floral organ abscission in Arabidopsis (5). It codes for a secreted precursor protein that requires processing to release the biologically active IDA peptide (6). The peptide binds to the extracellular leucine-rich repeat domain of the receptor-like kinases HAE (HAESA) (7, 8) and HSL2 (HAESA-LIKE2) (6, 9) and promotes association with co-receptors of the SERK (SOMATIC EMBRYOGENESIS RECEPTOR KINASE) family (8, 10). Intracellular signaling then activates genes that execute the abscission process (9, 11). Floral organs are shed when polygalacturonases and other hydrolytic enzymes degrade the pectin matrix of the middle lamella, resulting in cell separation in the abscission zone (1113). Here we identify the proteases required for IDA precursor (proIDA) processing and peptide maturation. We show that proIDA is processed by subtilisin-like proteases (subtilases, or SBTs) and that several functionally redundant SBTs contribute to IDA maturation in vivo.

Among the 56 SBTs in Arabidopsis, 54 are predicted to be located in the cell wall, where they appear to serve redundant functions, as indicated by the lack of obvious phenotypes for most of the single-gene loss-of-function mutants (14, 15). The majority of SBT genes are expressed in floral receptacles (figs. S1 and S2), where sepals, petals, and stamens attach to the plant body. With such a large number of SBTs potentially involved in the abscission process, existing methods aiming to impair gene function have poor prospects of success. We therefore developed a biochemical approach, addressing functional redundancy at the level of enzyme activity, by tissue-specific expression of a protease inhibitor. Two proteins from Phytophthora infestans [EPI1 (extracellular proteinase inhibitor 1) and EPI10] were chosen, comprising atypical Kazal-domains as specific inhibitors of SBTs but of no other serine proteases tested (16, 17) (figs. S3 and S4). The inhibitor sequences were codon-optimized for expression in plants and equipped with a plant signal peptide for efficient secretion. Transgenic plants expressing EPI1a (the atypical Kazal-domain of EPI1) or EPI10 in abscission zones under control of the IDA promoter retained their flower organs and thus phenocopied the abscission defect of the ida mutant (Fig. 1 and fig. S5). Abscission was restored when these plants were treated with a 14–amino acid (aa) peptide (mIDA) comprising the conserved PIPP motif that is required for receptor activation (8) (Fig. 2A). We conclude that SBT activity is required for abscission and, furthermore, that at least some SBTs act upstream of signal formation. Consistent with a role in IDA signaling, the expression of PGAZAT, a polygalacturonase gene controlled by the IDA signaling pathway and required for cell separation in abscission zones (12, 13), was impaired in EPI10-expressing transgenics and restored to wild-type (WT) levels after treatment with mIDA, whereas a longer peptide that still depends on processing for activation (eIDA) was inactive (Fig. 2, B and C).

Fig. 1 The shedding of floral organs depends on subtilisin-like proteinase (SBT) activity in abscission zones.

Arabidopsis Col-0 (WT) flowers complete abscission when they reach position P8 (the youngest flower after anthesis is in P1, older flowers are numbered consecutively). Transgenic plants expressing EPI1a or EPI10 under control of the IDA promoter retain their flower organs until fruit maturation.

Fig. 2 mIDA restores abscission and IDA target gene expression in IDAp::EPI10 transgenic lines.

(A) The delay of abscission (shown as percentage of flowers that shed their floral organs) observed in excised IDAp::EPI10 compared with WT inflorescences is overcome by mIDA (10 μM, fed through the cut stem; peptide sequence in Fig. 4A). Data represent the mean ± SD (error bars) for three independent transgenic lines, each analyzed in five experiments. (B) Reduced activity of the PGAZAT promoter in receptacles of IDAp::EPI10 flowers is restored to WT levels by treatment with mIDA. (C) Quantification of GUS activity (given as percentage of the WT control), confirming the histochemical results shown in (B). Peptides (mIDA and eIDA) were supplied at 10 μM. Data represent the mean of four experiments ± SEM (error bars).

To identify the precursor processing proteases, we selected 10 abscission-zone SBTs representing different SBT subfamilies and tested them for their ability to cleave the IDA precursor when coexpressed in Nicotiana benthamiana (fig. S6). Of the four proteases that affected IDA processing in this system, SBT4.12, SBT4.13, and SBT5.2 were partially purified from cell wall extracts of agro-infiltrated N. benthamiana plants and tested with a recombinant glutathione S-transferase (GST)–IDA fusion protein as the substrate. GST-IDA was processed by all three proteases and, in line with the abscission defect of the EPI1a- or EPI10-expressing transgenic plants, processing was sensitive to EPI10 inhibition (Fig. 3A). Low nanomolar concentrations of EPI1a and EPI10 were sufficient for half-maximal inhibition of SBT4.12, SBT4.13, and SBT5.2 (figs. S7 to S9). The dissociation constant (Kd) for the interaction of SBT4.13 with EPI1a was 23 ± 2 nM, whereas two binding events with Kd = 13 ± 2 nM and 0.8 ± 0.1 μM, respectively, were observed for the three-headed EPI10 inhibitor (Fig. 3B). The cumulative data—including (i) cleavage of the IDA precursor, (ii) coexpression with IDA in abscission zones, (iii) inhibition by the EPI inhibitors with inhibition and binding constants in the low nanomolar range, and (iv) the abscission defect of the EPI1a- or EPI10-expressing plants—support a role for SBT4.12, SBT4.13, and SBT5.2 in IDA processing and signal maturation.

Fig. 3 Processing of the IDA precursor by SBTs is inhibited by extracellular proteinase inhibitors (EPIs).

(A) Anti-GST immunoblot showing processing of recombinant GST-IDA upon addition of SBT4.12, SBT4.13, and SBT5.2 extracted from agro-infiltrated N. benthamiana plants (ev, empty vector control). Processing is inhibited in presence of EPI10. (B) Dissociation constants of SBT4.13–EPI inhibitor complexes determined by microscale thermophoresis. A biphasic saturation curve and two Kd values are observed for the three-headed EPI10 inhibitor (three experiments with independent preparations of the inhibitors). Error bars indicate SD.

As a candidate for the mature signal, each of the proteases released a 14-aa peptide from GST-IDA that was not formed in controls or when SBT activity was inhibited by EPI10 (fig. S10, A to C). The 14-aa peptide corresponds to part of the extended PIPP (ePIPP) motif (Fig. 4A) that is conserved between IDA and IDA-like proteins and contains the bioactive part of the protein (6). Within this region, a 12-aa peptide (PIPP) is minimally required for receptor (HAE) binding and co-receptor (SERK1) complex formation (8). The C-terminal Asn of PIPP is essential for receptor (HSL2) activation (18), and its carboxyl group needs to be free for binding (8). This Asn residue thus marks the C-terminal end of the endogenous IDA signal. Its N terminus is less clear, because N-terminal extension of PIPP by one or four amino acids does not alter receptor binding affinity (8, 18). We treated EPI10-expressing plants with the different peptides (Fig. 4A) to compare their abscission-inducing activity (Fig. 4B). In this bioassay, the 14-aa cleavage product was at least 10 times more active on a molar basis than PIPP or an N-terminally extended peptide (eIDA) (Fig. 4B). The residual activity of eIDA was lost when the peptide bond marking the N terminus of the 14-aa peptide (the Lys/Gly bond) was protected against proteolysis by N-methylation of the peptide backbone at this position (NMeIDA in Fig. 4B) (19). The highest abscission-inducing activity and the apparent requirement for processing of the Lys/Gly bond support the 14-aa peptide as the mature and bioactive form of IDA (mIDA).

Fig. 4 Identification of mIDA and processing-site confirmation in vivo.

(A) Representation of GST-IDA, processing sites, mIDA, and related peptides (not to scale). The lock icon marks the peptide bond protected against proteolysis by N-methylation. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr. (B) Dose-dependent abscission-inducing activity of the peptides is shown relative to water-treated IDAp::EPI10 and WT plants set to 0 and 100%, respectively [mean ± SD (error bars) for n = 6 biological replicates, including three independent transgenic lines]. (C) Sequence logo showing residues preferred by SBT4.13 upstream (P1 to P4) and downstream (P1′ to P6′) of the cleavage site (K/G), as analyzed by PICS (Proteomic Identification of Cleavage Sites). Asterisks highlight positions in which the residue that was most highly enriched over natural abundance matches the IDA precursor sequence. (D) Using Ala-substituted eIDA analogs as substrates of SBT4.13, P2-Pro and P4-Tyr were confirmed as the two most important residues for mIDA formation. Bars show the abundance of substrate (yellow) and products (red and gray; black represents the sum of all other peptides) in the reaction mixture as the percentage of all peptides identified by mass spectrometry. When P2-Pro was substituted (P2A), a miscleaved 15-aa peptide, rather than mIDA, was produced. (E) Genetic complementation of the ida abscission defect by IDAp::IDA-GFP and Ala-substituted mutants. Partial abscission refers to plants that retained flower organs in some positions older than P8 [mean ± SD (error bars) of 6 to 10 independent transformants].

In a genetic complementation experiment, we addressed the relevance of SBT-mediated precursor processing and mIDA formation for abscission in vivo. In contrast to WT IDA that is expected to restore abscission when expressed in the ida background, any precursor mutant that cannot be processed to produce bioactive mIDA will fail to do so. Failure of SBT-resistant mutants of the IDA precursor to complement the abscission defect of ida would thus confirm SBT-dependent processing as a prerequisite for IDA maturation. To generate IDA variants that are resistant to SBT cleavage, it is essential to know how these proteases recognize their substrates. In a proteomics approach [Proteomic Identification of Cleavage Sites (PICS)] (20), substrate libraries comprising more than 10,000 peptides were digested with SBT4.13 purified to homogeneity from agro-infiltrated N. benthamiana plants (fig. S11), and cleavage sites were identified by mass spectrometry. SBT4.13 was found to tolerate several different amino acids on both sides of the scissile bond (Fig. 4C); therefore, our first impression was that the protease is rather promiscuous with respect to cleavage site selection. However, with exception of the P1 position (the one immediately upstream of the cleavage site), the residues that were most highly preferred by SBT4.13 around the cleavage site of its peptide substrates were found in the corresponding positions flanking the processing site of the IDA precursor. Thus, substrate selectivity of SBT4.13 is not determined by a single amino acid in P1, but rather by an extended sequence motif including residues on both sides of the scissile bond. Pro and Tyr in P2 and P4, as well as Gly, Val, and Pro in P1′, P2′, and P5′, seemed particularly important (Fig. 4C). Using alanine-substituted synthetic peptides as substrates for SBT4.13, P2-Pro and P4-Tyr were confirmed as the two most important residues for substrate recognition on the nonprime side of the processing site (Fig. 4D). These substitutions are not part of mIDA and should thus not affect the bioactivity of the processing product.

To test whether recognition by subtilases and cleavage of the Lys/Gly bond are required for the generation of mIDA as the abscission signal in vivo, site-directed IDA mutants with Ala substitutions in P1, P2, and/or P4 were introduced into the ida background. Expression of the precursor variants in abscission zones was confirmed by the use of a C-terminally fused green fluorescent protein (GFP) tag (fig. S12). Consistent with the irrelevance of P1-Lys for cleavage site recognition (Fig. 4, C and D), the P1-Lys/Ala mutant (K/A in Fig. 4E) complemented the abscission defect of ida to almost the same extent as WT IDA-GFP. Substitution of Pro (P) in P2 in addition to P1-Lys (PK/A) reduced the ability to complement the mutant phenotype, and the P2-P4 double mutant (YP/A; Y, Tyr) was essentially inactive (Fig. 4E). Similarly, an ePIPP expression construct genetically complemented the ida mutant, and activity was impaired when Pro in P2 was substituted by Ala (P/A) (fig. S13).

Failure of the SBT-resistant precursor variants to restore abscission in the ida mutant confirms the IDA precursor as a physiological substrate of SBTs and indicates a role for SBTs in signal maturation. We conclude that precursor processing at the Lys/Gly bond is a prerequisite for the biogenesis of the abscission signal in Arabidopsis flowers, implicating the 14-aa peptide mIDA as the endogenous signal. Previous studies favored the 12-aa PIPP peptide (Fig. 4A) as the abscission signal (8, 18). We cannot exclude further trimming of the N terminus and the generation of PIPP by an unidentified aminopeptidase after initial cleavage of the Lys/Gly bond. However, considering the reduced activity of PIPP as compared with mIDA (Fig. 4B), this would decrease rather than increase signal intensity.

The close correlation observed between the amino acid residues required for the biogenesis of mIDA in vivo and for substrate recognition by SBT4.13 in vitro implicates SBT4.13 in the maturation process. However, abscission is normal in SBT4.13 single-gene loss-of-function mutants (fig. S5); therefore, SBT4.13 cannot be the only protease responsible. SBT4.12 and SBT5.2 are also involved, as they are coexpressed with IDA and SBT4.13 in abscission zones (fig. S2) and show the same cleavage specificity with respect to proIDA processing (fig. S10). The activity of several redundant subtilases is thus responsible for the biogenesis of the abscission signal.

Although AtSBT6.1, which is one of two intracellular SBTs and the ortholog of human site-1 protease, has previously been implicated in precursor processing (15, 2124), peptide hormone maturation by cognate SBTs was hitherto obscured by functional redundancy in the large SBT family. Using EPI inhibitors as a tool to overcome this functional redundancy, we demonstrated a role for SBTs in IDA maturation and abscission. Tissue-specific expression of EPI inhibitors will be instrumental for further analysis of SBTs in peptide hormone biogenesis. Tissue-specific expression of enzyme inhibitors may also be useful in other systems for the analysis of functionally redundant enzymes.

Supplementary Materials

Materials and Methods

Figs. S1 to S14

Table S1

Reference (25)

References and Notes

  1. Acknowledgments: We thank S. Kamoun (The Sainsbury Laboratory) for EPI1a and EPI10 expression constructs and R. Aalen (University of Oslo) for seeds of the ida mutant and the PGAZAT reporter line. We also thank them for fruitful discussions, as well as U. Glück-Behrens and J. Babo for technical assistance. This work was supported in part by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft) to A.Sc. (SCHA 591/3-2 and SCHA 591/4-1). The supplementary materials contain additional data.
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