The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

The peptide hormone PjCLE1 stimulates haustorium formation in the parasitic plant Phtheirospermum japonicum.

Phtheirospermum japonicum is a hemiparasitic plant of the Orobanchaceae, the largest family of parasitic plants. It extracts water and nutrients from other plants through haustoria along its roots. Haustoriogenesis, the formation of haustoria, is initiated by host-derived haustorium-inducing factors (HIFs). The first step in haustoriogenesis is the development of parasitically inactive protohaustoria. Here, we report that an endogenous peptide hormone, CLAVATA3/Embryo Surrounding Region 1 (PjCLE1), is sufficient to induce protohaustorium formation. PjCLE1 hyperactivated HIF-responses and caused prolific protohaustoria formation. PjCLE1 expression and activation by the subtilisin-type protease PjSBT1.2.3 occur in fully developed, mature haustoria, suggesting that PjCLE1 acts as an internal signal produced by mature haustoria to stimulate additional protohaustorium formation for effective extraction of resources from hosts. PjCLE1 is similar in sequence to CLEs regulating nodulation in legumes and part of a regulatory system for haustoria formation in parasitic plants.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation.

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

Sulfopeptide CLEL6 inhibits anthocyanin biosynthesis in Arabidopsis thaliana.

Posttranslationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small posttranslationally modified peptides are perceived by leucine-rich repeat receptor-like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibits anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is downregulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continues to act as negative regulators to limit pigment accumulation in the light.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis.

The hemiparasitic plant Phtheirospermum japonicum (Phtheirospermum) is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyze the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analyzed the subcellular localization, the enzymatic function and contribution to inducing hypertrophy by studying CRISPR-Cas9-induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed IPT activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a also was identified in the related weed Striga hermonthica. With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum-infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Processing and Formation of Bioactive CLE40 Peptide Are Controlled by Posttranslational Proline Hydroxylation.

Small posttranslationally modified signaling peptides are proteolytically derived from larger precursor proteins and subject to several additional steps of modification, including Pro hydroxylation, Hyp glycosylation, and/or Tyr sulfation. The processing proteases and the relevance of posttranslational modifications for peptide biogenesis and activity are largely unknown. In this study these questions were addressed for the Clavata3/Endosperm Surrounding Region (CLE) peptide CLE40, a peptide regulator of stem cell differentiation in the Arabidopsis (Arabidopsis thaliana) root meristem. We identify three subtilases (SBT1.4, SBT1.7, and SBT4.13) that cleave the CLE40 precursor redundantly at two sites. C-terminal processing releases the mature peptide from its precursor and is thus required for signal biogenesis. SBT-mediated cleavage at a second site within the mature peptide attenuates the signal. The second cleavage is prevented by Pro hydroxylation, resulting in the formation of mature and bioactive CLE40 in planta. Our data reveal a role for posttranslational modification by Pro hydroxylation in the regulation of CLE40 formation and activity.

Phytosulfokine (PSK) precursor processing by subtilase SBT3.8 and PSK signaling improve drought stress tolerance in Arabidopsis.

Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.

The Systemin Signaling Cascade As Derived from Time Course Analyses of the Systemin-responsive Phosphoproteome.

Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.

An OPR3-independent pathway uses 4,5-didehydrojasmonate for jasmonate synthesis.

Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3). Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role independent of JA-Ile and its receptor COI1; however, this hypothesis has been challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-didehydro-JA in wounded wild-type and opr3-3 plants. OPR2 was found to reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of OPR3, OPDA enters the ß-oxidation pathway to produce 4,5-ddh-JA as a direct precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA biosynthesis.

A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides.

The propeptides of subtilisin-like serine proteinases (subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana Sequence similarity and the shared ß-α-ß-ß-α-ß core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of Arabidopsis subtilase SBT4.13 with Kd and Ki values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of Arabidopsis SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13.

Phytaspase-mediated precursor processing and maturation of the wound hormone systemin.

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

From structure to function - a family portrait of plant subtilases.

Contents Summary 901 I. Introduction 901 II. Biochemistry and structure of plant SBTs 902 III. Phylogeny of plant SBTs and family organization 903 IV. Physiological roles of plant SBTs 905 V. Conclusions and outlook 911 Acknowledgements 912 References 912 SUMMARY: Subtilases (SBTs) are serine peptidases that are found in all three domains of life. As compared with homologs in other Eucarya, plant SBTs are more closely related to archaeal and bacterial SBTs, with which they share many biochemical and structural features. However, in the course of evolution, functional diversification led to the acquisition of novel, plant-specific functions, resulting in the present-day complexity of the plant SBT family. SBTs are much more numerous in plants than in any other organism, and include enzymes involved in general proteolysis as well as highly specific processing proteases. Most SBTs are targeted to the cell wall, where they contribute to the control of growth and development by regulating the properties of the cell wall and the activity of extracellular signaling molecules. Plant SBTs affect all stages of the life cycle as they contribute to embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell death, organ abscission, senescence, and plant responses to their biotic and abiotic environments. In this article we provide a comprehensive picture of SBT structure and function in plants.

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease.

Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall.

Dirigent Protein Mode of Action Revealed by the Crystal Structure of AtDIR6.

Dirigent proteins impart stereoselectivity to phenoxy radical coupling reactions in plants and, thus, play an essential role in the biosynthesis of biologically active natural products. This includes the regioselective and enantioselective coupling and subsequent cyclization of two coniferyl alcohol radicals to pinoresinol as the committed step of lignan biosynthesis. The reaction is controlled by dirigent proteins, which, depending on the species and protein, direct the reaction to either (+)- or (-)-pinoresinol. We present the crystal structure of the (-)-pinoresinol forming DIRIGENT PROTEIN6 (AtDIR6) from Arabidopsis (Arabidopsis thaliana) with data to 1.4 Å resolution. The structure shows AtDIR6 as an eight-stranded antiparallel ß-barrel that forms a trimer with spatially well-separated cavities for substrate binding. The binding cavities are two lobed, exhibiting two opposing pockets, each lined with a set of hydrophilic and potentially catalytic residues, including essential aspartic acids. These residues are conserved between (+) and (-)-pinoresinol-forming DIRs and required for activity. The structure supports a model in which two substrate radicals bind to each of the DIR monomers. With the aromatic rings fixed in the two pockets, the propionyl side chains face each other for radical-radical coupling, and stereoselectivity is determined by the exact positioning of the side chains. Extensive mutational analysis supports a previously unrecognized function for DIRs in catalyzing the cyclization of the bis-quinone methide reaction intermediate to yield (+)- or (-)-pinoresinol.

Cell Death Control by Matrix Metalloproteinases.

In contrast to mammalian matrix metalloproteinases (MMPs) that play important roles in the remodeling of the extracellular matrix in animals, the proteases responsible for dynamic modifications of the plant cell wall are largely unknown. A possible involvement of MMPs was addressed by cloning and functional characterization of Sl2-MMP and Sl3-MMP from tomato (Solanum lycopersicum). The two tomato MMPs were found to resemble mammalian homologs with respect to gelatinolytic activity, substrate preference for hydrophobic amino acids on both sides of the scissile bond, and catalytic properties. In transgenic tomato seedlings silenced for Sl2/3-MMP expression, necrotic lesions were observed at the base of the hypocotyl. Cell death initiated in the epidermis and proceeded to include outer cortical cell layers. In later developmental stages, necrosis spread, covering the entire stem and extending into the leaves of MMP-silenced plants. The subtilisin-like protease P69B was identified as a substrate of Sl2- and Sl3-MMP. P69B was shown to colocalize with Sl-MMPs in the apoplast of the tomato hypocotyl, it exhibited increased stability in transgenic plants silenced for Sl-MMP activity, and it was cleaved and inactivated by Sl-MMPs in vitro. The induction of cell death in Sl2/3-MMP-silenced plants depended on P69B, indicating that Sl2- and Sl3-MMP act upstream of P69B in an extracellular proteolytic cascade that contributes to the regulation of cell death in tomato.

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol.

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan5 strain to prevent hyperglycosylation. The two (His)6-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L-1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man5GlcNAc2 with, however, a significant contribution of Man4-10GlcNAc2. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.