Dimethyl allyl diphosphate (DMADP) is transformed to isoprene and pyrophosphate (PPi) thanks to the isoprene synthase. Is it better to:
- measure the concentration of isoprene synthase assuming similar rates among poplar species?
- measure the isoprene synthase gene expression (amount of mRNA)?
- measure the pool size of DMAPP within the leaf assuming a direct relationship of the substrate to the reaction?
- ...
Thank you in advance, RG community.
Hi,
Excellent question, no advice, but having worked on these biomolecules in the past am tempted to answer, and taking a stab at it, with some insights, which are worth considering:
1. Protein: let me discuss this first and get it out of way. There may be 'isoprene synthase' purified (buy or request a PI from a published paper) and one can generate antibodies or custom synthesize cheap polyclonal Abs for high through put ELISA-based quantification of synthase across species and tissue etc. Interferences from 'other' secondary metabolites in assays and purification can be challenging too.
'Assuming similar rates' could be flawed, and more than that "does protein amount correspond to their active/ inactive state (substrate availability, PTM modifications, phosphorylation, cellular location which is lost during maceration!)"? Clear, No. So, unsure if protein quantification is helpful.
2. mRNA: Gen expression based approaches have similar issues: compare if a protein has multiple isoforms, and splice variants, and nascent nucleolar vs. cytoplasmic (cistronic) mRNA vs truncated forms then what actually are you measuring? Are all transcripts translated ? No. So abundance of mRNA and their unexplainable normalization to total RNA prior to RTPCR or QPCR based quantification all lead to artefactual results. Also, why IPS measurement only and not expression one step upstream and downstream as well and then do a mathematics?
3. Metabolites: Just DMAPP pool is not helpful as it is easily converted (reversibly) into isoprene and PPi and is being co-shared for multiple other processes, i.e., protein prenylation/ ubiquitinylation?)? Also, measuring PPi would need LC/ spectrophotometer based assays, DMAPP as well; isoprene would need more GC-based volatile analysis; also would need differential extraction strategies (solvents, duration, methods). TLC-based assays for all the above are less reliable and sensitive. But, then PPi levels would be co-shared for zillions of other reactions within the cell. Also, given the variability in metabolite measurements, not a great choice to measure them. Enrichment would lead to loss of confidence in quantification.
Above all discuss the experimental/ strategic pitfalls.
Ques- Turn-over rates, which are faster? Proteins, transcripts or metabolites? Which are more reliable? Which is closer to the phenotype?
Cost and Time aspects: Very inexpensive for metabolites if all the analytical systems are in place and well optimized, as you buy some standards (or not!) and just spend for some solvents. But not a good idea to store samples/ transport/ share for analysis.
mRNA expression measurements can be high throughput and cheap too, can save samples infinitely, quicker and faster.
Protein based approaches can be time intensive and expensive too: raising antibodies, purification, custom synthesis etc. Stability and storage issues too.
Hope some of these help,
Thank you very much, Mr. Misra, for your thoughtful response. Thank you for taking the time to give your thoughts and share your experience. It is a very good starting point to continue discussing with other colleagues. All the best!
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