Periodic Reporting for period 1 - MultiPly (FMO1 Multifunctionality for improved Plant health)
Berichtszeitraum: 2023-09-01 bis 2025-08-31
‘MultiPly: FMO1 Multifunctionality for improved Plant health’
The project 'MultiPly' will investigate the extended role and evolution of the critically important FMO family in plants and examine the physiological mechanisms of how this enzyme can improve plant resilience under environmental stress conditions.
The long-term ambition of this project is to identify new and fundamental mechanisms of plant resistance for incorporation into crop breeding programs.
FMO1 is an essential enzyme for pathogen resistance in plants, and a valuable biotechnological target for crop health improvement. The importance of FMO1 for plant health has been recognised for more than 20 years, but its biochemical function in Arabidopsis was only recently determined: FMO1 catalyzes the N-hydroxylation of Pipecolic acid (Pip) to form N-hydroxy-pipecolic acid (N-Pip). N-Pip is the essential signalling molecule for Systemic Acquired Resistance (SAR) defense priming in plants (Figure 1). Following pathogen infection, a salicylic acid (SA) response cascade initiates N-Pip production at the site of infection, which is thereafter transported to distal tissues, signalling a new SA/SAR defense cascade in the uninfected tissue. FMO1 is therefore responsible for developing long-lasting and broad-spectrum disease resistance in plants and a potential target for metabolic engineering to improve disease resistance. In addition to that, studies on Barley FMO1 (HvFMO1) suggest that they possesses alternative functionality in vitro, catalysing the C-hydroxylation of indole to form indoxyl which then dimerizes to form the pigment indigo (Figure 2), highlighting potential diversification of function beyond pipecolic acid metabolism. These data suggest diversification of the FMO1 family in barley, both in terms of substrate specificity (indole vs pipecolic acid) and reaction mechanism (C-hydroxylation vs N-hydroxylation). It is possible that FMO1 functionality has diversified in other plant clades as well. Phylogenetic analyses further suggest duplication events in some eudicot species, pointing to evolutionary diversification within the FMO1 family (Figure 3). Together, these findings indicate that FMO1 may possess broader substrate specificity and roles in both biotic and abiotic stress responses. Understanding FMO1 multifunctionality is therefore highly relevant, not only to resolve fundamental questions of enzyme evolution, but also to identify new breeding targets for improving plant resilience and food security under climate change.
FMO1 is a flavin-containing monooxygenase (FMO) belonging to the N-hydroxylating (N-OX) clade. Plants also contain two other major FMO clades: the S-oxidizing (S-OX) FMOs and the YUCCA FMOs (involved in auxin biosynthesis). FMOs are notoriously difficult to purify, and comprehensive structure–function studies are rare.
To date, only a single plant FMO has been crystallized (an S-OX from garlic) and one YUCCA protein (AtYUCCA6) has been biochemically characterized. No structure–function studies have yet been reported for the plant FMO1 family, leaving major gaps in our understanding of their catalytic mechanisms, evolution, and functional diversification. Also to date, comprehensive substrate specificity analysis of FMO1 and other plant FMOs has not been performed.
Two key questions raised in MultiPly are:
1. Does FMO1 have additional biochemical functions or alternative substrates?
2. Is the N-Pip/SAR defence system conserved across all plants, or do alternative mechanisms exist?
The MultiPly project aims to addresses these questions by integrating biochemistry, structural biology, and evolutionary approaches (Figure 4).
Objectives of MultiPly:-
----------------------------
The project’s research objectives were organized in three work packages (WP1-WP3):
(1) Evolution and characterization of FMO1 sequence homology across the plant kingdom
(2) Characterization of plant FMO1s and their catalytic functionality in vitro and in vivo
(3) In planta characterization of SAR in barley (H. vulgare)
Thus by combining wet-lab enzymology with computational analyses, MultiPly aims to generate new insights into plant defense mechanisms, strengthen our molecular understanding of FMO1, and explore its potential as a target for engineering disease-resistant crops.
The project 'MultiPly' will investigate the extended role and evolution of the critically important FMO family in plants and examine the physiological mechanisms of how this enzyme can improve plant resilience under environmental stress conditions.
The long-term ambition of this project is to identify new and fundamental mechanisms of plant resistance for incorporation into crop breeding programs.
FMO1 is an essential enzyme for pathogen resistance in plants, and a valuable biotechnological target for crop health improvement. The importance of FMO1 for plant health has been recognised for more than 20 years, but its biochemical function in Arabidopsis was only recently determined: FMO1 catalyzes the N-hydroxylation of Pipecolic acid (Pip) to form N-hydroxy-pipecolic acid (N-Pip). N-Pip is the essential signalling molecule for Systemic Acquired Resistance (SAR) defense priming in plants (Figure 1). Following pathogen infection, a salicylic acid (SA) response cascade initiates N-Pip production at the site of infection, which is thereafter transported to distal tissues, signalling a new SA/SAR defense cascade in the uninfected tissue. FMO1 is therefore responsible for developing long-lasting and broad-spectrum disease resistance in plants and a potential target for metabolic engineering to improve disease resistance. In addition to that, studies on Barley FMO1 (HvFMO1) suggest that they possesses alternative functionality in vitro, catalysing the C-hydroxylation of indole to form indoxyl which then dimerizes to form the pigment indigo (Figure 2), highlighting potential diversification of function beyond pipecolic acid metabolism. These data suggest diversification of the FMO1 family in barley, both in terms of substrate specificity (indole vs pipecolic acid) and reaction mechanism (C-hydroxylation vs N-hydroxylation). It is possible that FMO1 functionality has diversified in other plant clades as well. Phylogenetic analyses further suggest duplication events in some eudicot species, pointing to evolutionary diversification within the FMO1 family (Figure 3). Together, these findings indicate that FMO1 may possess broader substrate specificity and roles in both biotic and abiotic stress responses. Understanding FMO1 multifunctionality is therefore highly relevant, not only to resolve fundamental questions of enzyme evolution, but also to identify new breeding targets for improving plant resilience and food security under climate change.
FMO1 is a flavin-containing monooxygenase (FMO) belonging to the N-hydroxylating (N-OX) clade. Plants also contain two other major FMO clades: the S-oxidizing (S-OX) FMOs and the YUCCA FMOs (involved in auxin biosynthesis). FMOs are notoriously difficult to purify, and comprehensive structure–function studies are rare.
To date, only a single plant FMO has been crystallized (an S-OX from garlic) and one YUCCA protein (AtYUCCA6) has been biochemically characterized. No structure–function studies have yet been reported for the plant FMO1 family, leaving major gaps in our understanding of their catalytic mechanisms, evolution, and functional diversification. Also to date, comprehensive substrate specificity analysis of FMO1 and other plant FMOs has not been performed.
Two key questions raised in MultiPly are:
1. Does FMO1 have additional biochemical functions or alternative substrates?
2. Is the N-Pip/SAR defence system conserved across all plants, or do alternative mechanisms exist?
The MultiPly project aims to addresses these questions by integrating biochemistry, structural biology, and evolutionary approaches (Figure 4).
Objectives of MultiPly:-
----------------------------
The project’s research objectives were organized in three work packages (WP1-WP3):
(1) Evolution and characterization of FMO1 sequence homology across the plant kingdom
(2) Characterization of plant FMO1s and their catalytic functionality in vitro and in vivo
(3) In planta characterization of SAR in barley (H. vulgare)
Thus by combining wet-lab enzymology with computational analyses, MultiPly aims to generate new insights into plant defense mechanisms, strengthen our molecular understanding of FMO1, and explore its potential as a target for engineering disease-resistant crops.
Technical and scientific part only
Work Plan 1: Evolution and characterisation of FMO1 sequence homology across the plant kingdom
-------------------------------------------------------------------------------------------------------------------------
Work performed and plans:-
1. In silico analysis to compare the evolutionary diversity of FMO1 and FMO-like protein in the green lineage
To explore FMO diversification and evolution across the plant kingdom, I constructed phylogenetic trees (Figure 5). FMO sequences were retrieved from the available genomic, transcriptomic, and gene-expression databases (e.g. GenBank, Genevestigator, IPK, 1000 Plants, Phytozome, PlantGDB), and FMO1 expression patterns—especially in response to environmental stimuli—were compared to select FMOs (Figure 6) for further biochemical characterisation in WP2. Conserved motifs and sequence logos were identified using SMART and MEME Suite, and in silico modelling and docking were performed in AlphaFold 3 version( Figure 7).
Plans for the next period:-
1. Finalize the selected 10-11 genes for further studies by checking all in silico characteristic features (including solubility predictions).
2. Integrate AlphaFold models
3. Prepare methodology and plans for next period
Work Plan 2: Characterisation of plant FMOs and their catalytic functionality in vitro and in vivo
--------------------------------------------------------------------------------------------------------------------
Work performed:-
1. For the selected 11 FMO1 and FMO1-like genes established in vitro (E.coli) expression screening (BL21 DE3, Rosetta, Nico21 pRARE2) with pET and pNIC28-Bsa4 constructs, guided by NetSolP insilico solubility prediction studies.
2. For the selected FMO1s- after multiple screening trials, Nico21 pRARE2 was selected as the best expression system. Fresh LB-agar plate containing 50 µg/mL kanamycin and 25 µg/mL chloramphenicol were used to grow the colonies.
3. After proper transformation of FMO1s in Nico21 pRARE2, the cells were plated on LB agar plates with appropriate antibiotics and incubated overnight. The plates were closed observed for any color formation (Figure 8). For HvFMO1, blue color colonies were observed immediately, however for SbFMO1 and HvFMO27, blue colonies were observed after 3-4 days. For other selected FMOs no color was observed. Further, to exclude vector-specific artefacts, we re-cloned and re-screened all FMO constructs in the same expression backbone; the color-forming variants (HvFMO1, SbFMO1) reproduced the indigo/indirubin phenotype, while empty-vector controls remained negative.
3. Small-scale expression and purification trails (10 ml culture) were used for the optimization. After multiple trails based on temperature (range of 15 to 37 degree Celsius), and timing (4-24 hours), 18 degree, overnight incubation was further used for large scale cultures. Buffers were also optimized.
4. Large scale expression and purification (1Litre), were done once the conditions and buffers are optimized. For large scale, to get seed culture, inoculated fresh colony in 50 mL TB medium supplemented with 50 µg/mL kanamycin and 25 µg/mL chloramphenicol , and grown at 37 °C(250 rpm). Furthermore, for each litre of TB medium (supplemented with kanamycin and chloramphenicol) with 20 mL bacterial seed culture were used. Pre-induction cultures were incubated (37 °C, 250 rpm) until OD₆₀₀ = 1.2–1.5. Subsequently, protein expression was induced with 0.5 mM IPTG, the culture was incubated O/N at 18 °C (150 rpm, ∼16–18 hours) and the E. coli cells were harvested by centrifuging in a SORVALL LYNX 6000 centrifuge (4 °C, 5000 × g for 10-15 min). E. coli cells were resuspended in optimized lysis buffer (100 mM Tris pH 7.5 300 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.04 mg/mL lysozyme, protease inhibitor (1 tablet/50 mL buffer), Benzonase (Sigma-Aldrich), and NP-40 (0.05%), 5% Glycerol). Lysis buffer usage -5 mL/gram of pelleted cells. The cells were resuspended and incubated on ice for 30–60 min (cold room). Just before sonication added FAD (100µM) as it is cofactor dependent protein and also to maintain stability. The cells were disrupted using a Branson Ultrasonics Sonifier 250 on ice, with a very high energy microtip probe (Output control: 5, amplitude: 40%, 5 sec on, 25 sec off, 10 min). The lysate was then centrifuged in a SORVALL LYNX 6000 centrifuge (10,000 × g, 45 min), and the supernatant (crude fraction) was collected and used for the crude enzyme assay. Ni-NTA Affinity purification were also performed to get purified proteins (Buffer details: Binding buffer- 50mM Tris pH 7.5 150mM NaCl, 0.5mM DTT, 10mM imidazole, 5% Glycerol); Wash Buffer - 50mM Tris, 150mM NaCl, 10mM Imidazole, 0.5mM DTT, 5% Glycerol; Elution Buffer - 50mM Tris, 150mM NaCl, 100mM Imidazole, 0.5mM DTT, 5% Glycerol). PAGE profile for purified fractions are Given in Figure 9.
4. Substrate specificity screening- Using the crude enzyme as well as purified (based on availability), different substrates (i.e. pipecolic acid, indole, proline, serotonin, Tryptophan, tryptamine etc.) were screened and product formation were analysed by LC-MS.
5. Coenzyme selectivity (NADPH vs. NADH) were also studied (Figure 9).
Major training (Secondment) during this period:-
---------------------------------------------------------
I got a great opportunity to spend time in Molecular Enzymology Group, University of Groningen, Netherlands. It was an excellent training, and I could get expert guidance from the group and also could involve in multiple discussion with group members. Based on this international research secondment, I could further develop my skills within FMO enzyme biology and also got extensive network within academic research and industry. I mainly studied an FMO1-like enzyme from Barley (HvFMO7). Along with HvFMO27, HvFMO1, and SbFMO1 were also tried as controls.
Work plan 3 : In planta characterization of SAR in barley (H. vulgare)
------------------------------------------------------------------------------------
This objective is to determine whether alternative SAR-like mechanisms exist in barley beyond the classical FMO1–NHP pathway, and evaluate VOC emissions during infection.
Work performed (this period):-
1. For in planta SAR characterization: Set up barley growth pilot studies (cv. Golden Promise / RGT Planet)
2. Generated preliminary evidence for an alternative SAR-like route in Barley linked to tryptamine oxidation (Tryptamine → 2-oxotryptamine (2OT)) by FMO1-like enzyme named as HvFMO27 in my studies (supported by the WP2 enzyme data).
3. Observed HvFMO27 upregulation under pathogen stress, suggesting a defence role.
For VOC assessment:
1. Had multiple planning/discussion meetings (online and offline) with collaboration partners and had discussion with technical persons too . Checked for VOC test chambers and plant handling. However due to the biological and technical complexity of the in planta VOC setup, substantial optimization requirement for this growth setup, we decided to focus more on the alternative SAR route found in Barley (2OT production). Pathogen handling was another bottleneck- as pathogen work was not permitted in the partner facility, and there were logistical constraints that prevented relocating the PTR–TOF–MS instrument to my laboratory. As a result, the original WP3 plan was reframed little to prioritize the in vitro enzymatic studies and functional characterization of the barley FMO1 paralog, HvFMO27. This deviation could result rapid progress in project and obtained robust results, including the identification of tryptamine oxidation to 2-oxotryptamine (2OT) as an alternate defence pathway in barley. The outcomes shared as a preprint and have been submitted to Plant Physiology, ensuring that the project continued to deliver scientific impact despite infrastructure limitations.
Achievements-
1. Successfully did the phylogeny and bioinformatics analysis of FMO/FMO1 across the green lineage (motifs identified; AlphaFold models generated to guide experiments).
2. Selected FMOs were successfully expressed in E. coli (NiCo21 pRARE2), and could achieve purification for many (HvFMO1, SbFMO1, HvFMO27).
3. Could establish an active collaboration and could spend time for the secondment, internationally (advanced skills gained in expression/purification of flavoproteins, assay design, troubleshooting, data analysis; expanded academic networking).
4. Had multiple collaborative Zoom and direct meetings with Prof. Riikka Rinnan for VOC workflow; could not establish the system within the timeframe due to logistics/pathogen constraints—efforts continued with alternative plans- But established a potential network.
5. Confirmed NADPH preference
6. Could develop and optimise enzymatic assay and LC–MS sample preparation to detect 2-oxotryptamine (2OT), Tryptamine oxidation product via HvFMO27 activity (key mechanistic finding for alternative SAR-like signalling).
7. Could set up rapid colorimetric screens: indigo (HvFMO1, HvFMO27) and indirubin (SbFMO1); re-cloning into the same vector reproduced color, ruling out vector artefacts.
8. Completed small- to large-scale expression workflows with cofactor-stabilized buffers and optimised induction/temperature, and storage of enzymes.
9. Established crude and purified enzymatic assays.
10. Disseminated results effectively: WP1- In silico preliminary work is shared in preprint server and later accepted in The Plant Journal. Further a joint WP2–WP3 study is available in BioRxiv and submitted to Plant Physiology; 2 poster presentations were done at two conferences (EMBO and SPSS).
11. Could strengthen transferable competencies through the secondment and collaborations: experimental design, method development, project coordination, network building, and cross-disciplinary communication.
Work Plan 1: Evolution and characterisation of FMO1 sequence homology across the plant kingdom
-------------------------------------------------------------------------------------------------------------------------
Work performed and plans:-
1. In silico analysis to compare the evolutionary diversity of FMO1 and FMO-like protein in the green lineage
To explore FMO diversification and evolution across the plant kingdom, I constructed phylogenetic trees (Figure 5). FMO sequences were retrieved from the available genomic, transcriptomic, and gene-expression databases (e.g. GenBank, Genevestigator, IPK, 1000 Plants, Phytozome, PlantGDB), and FMO1 expression patterns—especially in response to environmental stimuli—were compared to select FMOs (Figure 6) for further biochemical characterisation in WP2. Conserved motifs and sequence logos were identified using SMART and MEME Suite, and in silico modelling and docking were performed in AlphaFold 3 version( Figure 7).
Plans for the next period:-
1. Finalize the selected 10-11 genes for further studies by checking all in silico characteristic features (including solubility predictions).
2. Integrate AlphaFold models
3. Prepare methodology and plans for next period
Work Plan 2: Characterisation of plant FMOs and their catalytic functionality in vitro and in vivo
--------------------------------------------------------------------------------------------------------------------
Work performed:-
1. For the selected 11 FMO1 and FMO1-like genes established in vitro (E.coli) expression screening (BL21 DE3, Rosetta, Nico21 pRARE2) with pET and pNIC28-Bsa4 constructs, guided by NetSolP insilico solubility prediction studies.
2. For the selected FMO1s- after multiple screening trials, Nico21 pRARE2 was selected as the best expression system. Fresh LB-agar plate containing 50 µg/mL kanamycin and 25 µg/mL chloramphenicol were used to grow the colonies.
3. After proper transformation of FMO1s in Nico21 pRARE2, the cells were plated on LB agar plates with appropriate antibiotics and incubated overnight. The plates were closed observed for any color formation (Figure 8). For HvFMO1, blue color colonies were observed immediately, however for SbFMO1 and HvFMO27, blue colonies were observed after 3-4 days. For other selected FMOs no color was observed. Further, to exclude vector-specific artefacts, we re-cloned and re-screened all FMO constructs in the same expression backbone; the color-forming variants (HvFMO1, SbFMO1) reproduced the indigo/indirubin phenotype, while empty-vector controls remained negative.
3. Small-scale expression and purification trails (10 ml culture) were used for the optimization. After multiple trails based on temperature (range of 15 to 37 degree Celsius), and timing (4-24 hours), 18 degree, overnight incubation was further used for large scale cultures. Buffers were also optimized.
4. Large scale expression and purification (1Litre), were done once the conditions and buffers are optimized. For large scale, to get seed culture, inoculated fresh colony in 50 mL TB medium supplemented with 50 µg/mL kanamycin and 25 µg/mL chloramphenicol , and grown at 37 °C(250 rpm). Furthermore, for each litre of TB medium (supplemented with kanamycin and chloramphenicol) with 20 mL bacterial seed culture were used. Pre-induction cultures were incubated (37 °C, 250 rpm) until OD₆₀₀ = 1.2–1.5. Subsequently, protein expression was induced with 0.5 mM IPTG, the culture was incubated O/N at 18 °C (150 rpm, ∼16–18 hours) and the E. coli cells were harvested by centrifuging in a SORVALL LYNX 6000 centrifuge (4 °C, 5000 × g for 10-15 min). E. coli cells were resuspended in optimized lysis buffer (100 mM Tris pH 7.5 300 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.04 mg/mL lysozyme, protease inhibitor (1 tablet/50 mL buffer), Benzonase (Sigma-Aldrich), and NP-40 (0.05%), 5% Glycerol). Lysis buffer usage -5 mL/gram of pelleted cells. The cells were resuspended and incubated on ice for 30–60 min (cold room). Just before sonication added FAD (100µM) as it is cofactor dependent protein and also to maintain stability. The cells were disrupted using a Branson Ultrasonics Sonifier 250 on ice, with a very high energy microtip probe (Output control: 5, amplitude: 40%, 5 sec on, 25 sec off, 10 min). The lysate was then centrifuged in a SORVALL LYNX 6000 centrifuge (10,000 × g, 45 min), and the supernatant (crude fraction) was collected and used for the crude enzyme assay. Ni-NTA Affinity purification were also performed to get purified proteins (Buffer details: Binding buffer- 50mM Tris pH 7.5 150mM NaCl, 0.5mM DTT, 10mM imidazole, 5% Glycerol); Wash Buffer - 50mM Tris, 150mM NaCl, 10mM Imidazole, 0.5mM DTT, 5% Glycerol; Elution Buffer - 50mM Tris, 150mM NaCl, 100mM Imidazole, 0.5mM DTT, 5% Glycerol). PAGE profile for purified fractions are Given in Figure 9.
4. Substrate specificity screening- Using the crude enzyme as well as purified (based on availability), different substrates (i.e. pipecolic acid, indole, proline, serotonin, Tryptophan, tryptamine etc.) were screened and product formation were analysed by LC-MS.
5. Coenzyme selectivity (NADPH vs. NADH) were also studied (Figure 9).
Major training (Secondment) during this period:-
---------------------------------------------------------
I got a great opportunity to spend time in Molecular Enzymology Group, University of Groningen, Netherlands. It was an excellent training, and I could get expert guidance from the group and also could involve in multiple discussion with group members. Based on this international research secondment, I could further develop my skills within FMO enzyme biology and also got extensive network within academic research and industry. I mainly studied an FMO1-like enzyme from Barley (HvFMO7). Along with HvFMO27, HvFMO1, and SbFMO1 were also tried as controls.
Work plan 3 : In planta characterization of SAR in barley (H. vulgare)
------------------------------------------------------------------------------------
This objective is to determine whether alternative SAR-like mechanisms exist in barley beyond the classical FMO1–NHP pathway, and evaluate VOC emissions during infection.
Work performed (this period):-
1. For in planta SAR characterization: Set up barley growth pilot studies (cv. Golden Promise / RGT Planet)
2. Generated preliminary evidence for an alternative SAR-like route in Barley linked to tryptamine oxidation (Tryptamine → 2-oxotryptamine (2OT)) by FMO1-like enzyme named as HvFMO27 in my studies (supported by the WP2 enzyme data).
3. Observed HvFMO27 upregulation under pathogen stress, suggesting a defence role.
For VOC assessment:
1. Had multiple planning/discussion meetings (online and offline) with collaboration partners and had discussion with technical persons too . Checked for VOC test chambers and plant handling. However due to the biological and technical complexity of the in planta VOC setup, substantial optimization requirement for this growth setup, we decided to focus more on the alternative SAR route found in Barley (2OT production). Pathogen handling was another bottleneck- as pathogen work was not permitted in the partner facility, and there were logistical constraints that prevented relocating the PTR–TOF–MS instrument to my laboratory. As a result, the original WP3 plan was reframed little to prioritize the in vitro enzymatic studies and functional characterization of the barley FMO1 paralog, HvFMO27. This deviation could result rapid progress in project and obtained robust results, including the identification of tryptamine oxidation to 2-oxotryptamine (2OT) as an alternate defence pathway in barley. The outcomes shared as a preprint and have been submitted to Plant Physiology, ensuring that the project continued to deliver scientific impact despite infrastructure limitations.
Achievements-
1. Successfully did the phylogeny and bioinformatics analysis of FMO/FMO1 across the green lineage (motifs identified; AlphaFold models generated to guide experiments).
2. Selected FMOs were successfully expressed in E. coli (NiCo21 pRARE2), and could achieve purification for many (HvFMO1, SbFMO1, HvFMO27).
3. Could establish an active collaboration and could spend time for the secondment, internationally (advanced skills gained in expression/purification of flavoproteins, assay design, troubleshooting, data analysis; expanded academic networking).
4. Had multiple collaborative Zoom and direct meetings with Prof. Riikka Rinnan for VOC workflow; could not establish the system within the timeframe due to logistics/pathogen constraints—efforts continued with alternative plans- But established a potential network.
5. Confirmed NADPH preference
6. Could develop and optimise enzymatic assay and LC–MS sample preparation to detect 2-oxotryptamine (2OT), Tryptamine oxidation product via HvFMO27 activity (key mechanistic finding for alternative SAR-like signalling).
7. Could set up rapid colorimetric screens: indigo (HvFMO1, HvFMO27) and indirubin (SbFMO1); re-cloning into the same vector reproduced color, ruling out vector artefacts.
8. Completed small- to large-scale expression workflows with cofactor-stabilized buffers and optimised induction/temperature, and storage of enzymes.
9. Established crude and purified enzymatic assays.
10. Disseminated results effectively: WP1- In silico preliminary work is shared in preprint server and later accepted in The Plant Journal. Further a joint WP2–WP3 study is available in BioRxiv and submitted to Plant Physiology; 2 poster presentations were done at two conferences (EMBO and SPSS).
11. Could strengthen transferable competencies through the secondment and collaborations: experimental design, method development, project coordination, network building, and cross-disciplinary communication.
During the MultiPly project, we achieved some results that advance the field of FMO enzyme multifunctionality, structural bioinformatics, and plant biochemistry beyond the current state of knowledge
A. Novel Functional Insights:-
1. Provided a classification schema and naming system for class-B FMOs (plant YUCCA enzymes), and details about FMO1 motif variation among various FMOs (Manuscript in preprint server)
2. We delivered the first integrated structural–evolutionary analysis of plant FMOs, mapping conserved motifs/active-site determinants and lineage variation, and linking these features to predicted substrate access and function.
These findings go beyond the existing knowledge, where plant FMOs had previously been studied in isolation, with limited structural and evolutionary characterization.
B. Integration of Wet- and Dry-Lab Approaches for FMO studies
By combining recombinant protein expression, purification, enzyme assays, LC–MS-based assays, and computational design, we established a transferable workflow for FMO1 studies in plants.
This approach is transferable to other enzyme families relevant for crop improvement, sustainable agriculture, sustainable biocatalysis and biosensor development.
C. We established a transferable pipeline that couples heterologous expression/purification, in vivo color assays (indigo/halo-indigo), and LC–MS enzyme assays with computational modelling. This workflow enabled identification of tryptamine → 2-oxotryptamine (2OT) activity (barley FMO1 paralog, 2OTS) and NADPH preference, and is readily reusable for enzyme families.
C. Computational Pipelines and Data Resources
Developed pipelines for phylogenetic profiling, domain annotation, and docking simulations that can be applied to other uncharacterised plant enzymes.
Results contribute to FAIR data principles, and also open novel directions for protein design in plant biotechnology.
A. Novel Functional Insights:-
1. Provided a classification schema and naming system for class-B FMOs (plant YUCCA enzymes), and details about FMO1 motif variation among various FMOs (Manuscript in preprint server)
2. We delivered the first integrated structural–evolutionary analysis of plant FMOs, mapping conserved motifs/active-site determinants and lineage variation, and linking these features to predicted substrate access and function.
These findings go beyond the existing knowledge, where plant FMOs had previously been studied in isolation, with limited structural and evolutionary characterization.
B. Integration of Wet- and Dry-Lab Approaches for FMO studies
By combining recombinant protein expression, purification, enzyme assays, LC–MS-based assays, and computational design, we established a transferable workflow for FMO1 studies in plants.
This approach is transferable to other enzyme families relevant for crop improvement, sustainable agriculture, sustainable biocatalysis and biosensor development.
C. We established a transferable pipeline that couples heterologous expression/purification, in vivo color assays (indigo/halo-indigo), and LC–MS enzyme assays with computational modelling. This workflow enabled identification of tryptamine → 2-oxotryptamine (2OT) activity (barley FMO1 paralog, 2OTS) and NADPH preference, and is readily reusable for enzyme families.
C. Computational Pipelines and Data Resources
Developed pipelines for phylogenetic profiling, domain annotation, and docking simulations that can be applied to other uncharacterised plant enzymes.
Results contribute to FAIR data principles, and also open novel directions for protein design in plant biotechnology.