Technical and scientific part only
Work Plan 1: Evolution and characterisation of FMO1 sequence homology across the plant kingdom
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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
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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:-
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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)
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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.
