Ok... maybe I'm being simple here but, GPP, FGP and GGPP are cellular intermediates which are rapidly metabolised. Any intracellular build up in these would infer inefficient transformation to terpenoids. There is some evidence from studies on indole alkaloid biosynthesis (label scrambling) that terpenoid precursors can be recycled. The corresponding cations are (still hypothetical) species produced in the cyclase enzymes. I think the best answer might be to focus on determination of the levels of the final products of your system. Why not set up an HPLC assay to determine the levels of the final products?

Ander,

are you interested in the induced production/accumulation of terpenes or the differential activity of certain biosynthetic steps? A metabolite level analysis (GCMS, HPLC) would be the easiest to develop tool to demonstrate changes in terpene production and would cover a broad range of potential products.

I think Rayko has focussed on the problem - are you trying to determine gene expression, enzyme activity or metabolite production?

Dear all, thank you very much for your answers. I'm sorry if I did not expose my question clearly, i'm new at this topic.

We have studied and we are studying the terpene concentration (mono, sesqui, di) and emission (mono, sesqui) of cypress atacked by a fungal pathogen, and we do that by GC-MS. The next step is relating these concentrations/emissions we have to the indcution, which I understand that can be measured by enzyme activity or by gene expression. As it is the first time, we wanted to have a global vision, that is, not the induction of a single monoterpene, but all monoterpenes. I've seen that sometimes it's not easy to relate the synthase expression to the final product that you find (e.g. higher expression under attack, but no difference in concentration) so we thought it was nice to begin taking an overview, which is maybe less prone to this problems. I guess that gene expression is the best way, but I'm afraid it's not possible to have this global vision, but a vision of a specific terpene synthase (apart from that, there are no primers avilable for cypress). That is what I'm asking: Is there a method, with gene expression, to look at the mono in general, the sesqui in general and diterpenes in general that have been induced? or this can only be done by enzyme activity?

PD: Robert Baxter, I was precisely afraid of that, thanks!

Cypress.... OMG.... produces a wide range of terpenoids, as far as I know, there are at least six major monoterpenes in the leaves. This would NOT be my choice of a system to study. Are you doing this with tissue culture?

So .... if it were my project ...I'd try to limit it to realistic objectives.

Questions (given the fact that you don't have an identifyable gene ):-

1. What are the changes in metabolite production under stress?

(i.e. is there a change in metabolism from unoxidated terpenes (eg limonene) to oxidated ones; or from mono to diterperpenes?). This is really an HPLC problem.

2. Does this stress change levels of GPP and FGP levels in the cells? (see Dr Simonsen's comments on this).

It really depends on whether you are looking at a tissue cell culture or a plant.

best wishes

Haha, I found around 50 different terpenes in the leaves, and around 70 in the phloem of these trees (not cultured cells). That's why I'm talking about overviews, though I could focus on some specific compounds (but I have no synthase!). May be hard, but the article I attached is for pines, which is not simpler... so I'm sure it can be done. Thanks for caring.

Actual quantification by GC/MS seems stil realistic for me. But another idea is RNA-seq and compare putative synthase before and after biotic attack.

Ok... I've read the Litvak and Monson paper. It was published sixteen years ago. Technology has moved on. I'm sure you could repeat their work on Cypress ... but would it add anything meaningful to science?

Hi Robert. My intention is to do it with "modern" technology, otherwise I wouldn't have asked the question! but if I'm not able to find a modern way (gene expression) to measure the mt, st and dt in general, I may do it the old way, or maybe study a specific synthase, though that is like a whole new project. I will talk a bit to Dr. Simonsen about the possibilities. I consider that, even if it was only enzyme activity, it is meaningful for science, since nobody has ever studied how the cyclases eznyme activity of a cypress works. We want to relate it to the terpene contents.. contents and gene expression/enzyme activity do not always match, so it is interesting to have more information, specially of a genus that remains unstudied. Cheers!

Interesting question