Hi,
I am working on sulfate metabolism wit Arabidopsis plant and its GDC knock-down mutant. I have a huge list of RNA-Seq results of genes involved in sulfur reduction and assimilation. Some genes as ATPS, APR2, SiR, OASB, gama-ECS are very slight upregulated in mutant comparing with wild type (up to 1 fold or 1-1.5 fold), some significantly some of them not. Beside that I have very high content of Cysteine, glutathione, but at the same time reduced flux of sulfate in this pools while content of total proteins remain almost at the same level for wild type and mutant.
Does it possible, in my case, that even small upregulation in gene expression of some genes from sulfate reduction and assimilation pathway can lead to even 4 fold higher accumulation of Cys and 8 fold higher accumulation of glutathione in mutant comparing with wild type?
I am trying to explain if this high contents of Cys and glutathione together with low flux can be explained by changes in gene transcription.
Thank you very much for your help!
Sladja
I'm not too savvy in those metabolites and pathways, but could it be that the genes regulating the flux away from those metabolites are downregulated?
I've done some work on lipid synthesis pathways in algae for my PhD. We found that some lipids were accumulating in the middle of the pathway, despite the enzyme leading to them not being upregulated. We thought one potential explanation could be that the next enzyme in the pathway was downregulated, leading to the accumulation. This was based on qPCR, so we did not have data on all the enzymes involved unfortunately.
Dear Sladjana Samuilov ,
very interesting question! But your question is very relevant and often overlooked: expression level is not related to flux. For a pathway modification to work, the limiting step should be identified. From the observation that cysteine and glutathionine levels are elevated, one can draw conclusions about which step in the sulphur metabolism is limiting. Just look up the sulphur pathway and you will see which enzyme may be limiting. Try overexpressing it and then look again. I am sure that you will get a very nice experiment;)
kind regards, Harrie Verhoeven.
As a complement to what have been said above, I think that accumulation of several small changes in expression of different enzymes / proteins in a pathway can lead to a significant output in terms of flux. That's partly what Gene Set Analysis (http://www.broadinstitute.org/gsea) tries to decipher by looking not only to single genes variations, but also to genes which are linked by a function, a pathway, a response to some stimuli... I don't know if Gene Sets are available for Arabidopsis, but they can be easily created if you know your pathway. Then you'll see if the pathway is significantly enriched amongst upregulated genes, or if it's just a coincidence / feeling. Good luck!
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