Thank yu for the suggestion Stafano. However, at the moment I am not interested as much in the response per se, but rather in how is the response triggered.

That is, in order to modulate gene expression and to exert physiological effect, the molecule must be perceived, the signal must be transmitted into the nucleus and there it must change the gene expression. Yet it doesn't bind to canonical cytokinin receptors. So what would be the easiest way to determine the sensing protein?

Thank you for the suggestions.

But even the oxidation status/pH/etc. must be affected somehow, mustn't they? How do they change in response to a particular compound? Or is it that the compound changes it directly, e.g. an acid changes the pH, while some redox compound would change the oxidation status?

Well, the compound doesn't seem to posses any such qualities to change significantly the environment. Moreover, the response seem to be specific, once it is both tZ9G and tZ7G (different position of Glc on the Ade), once it is both tZ9G and iP9G (missing hydroxyl on the side chain).

Even if it interacted with any channel or pump, that would be great. I'll look into the PROMIS you shared.

Stefano D'Alessandro I've been thinking about that affinity purification as you suggested. The problem is, that the side chain (making the compound a cytokinin) and the glucose (as I'm not interested in free base) are both on opposite sides of the molecule, so it would be probably difficult to design such compound, which would retain both properties and carry also the linker.

The radioactivity showed no conversion. Nothing that could be ascribed to cytokinins, maybe some degradation, but that's not really surprising, because the glucosides shall be substrates of the degrading enzyme.

No, I have not tested it on mutant plants, because right now I'm working with oat. I'm thinking about switching to maize, because that would be at least little bit easier. The problem is, that the effect on Arabidopsis is somewhat weird. But I'll have to check on it in more detail.

Hi Tomas,

Ho can you know that the physiological effect you are observing comes from the exogenously applied CK glucosides and is not caused by the increased level of CK free bases? Even if plants don't have receptors to actively bind 7- or 9- glucosides, they must definitely sense their presence and maybe it is possible that in order to keep CK homeostasis on a stable level plant will produce additional free base CKs that in turn cause the observed physiological effect? Are the glucosides you are using present in control samples at any level? I think that the presence of any naturally occurring biological compound that differs from its physiological levels will trigger plant response even if the compound itself doesn't respond to any CK receptors. To me it looks like your interference with CK homeostasis might be the reason that causes the cycle of the observed changes (e.g. plant doesn't want to slow down the metabolism as requested by the increased gluc levels so it increases the biosynthesis of the active CK forms to counteract the effect cased by the non-active glucosides).

Hi Anna Kisiala ,

you're totally right. There is increase of free base cytokinins and although it appeared to originate through hydrolysis of the glucosides, the current data say otherwise.

But even in such case, the plant has to somehow sense that there is inbalance and too much of cytokinin glucosides. How would that be?

Hi Tomas. My original thinking was that the plant senses the presence of glucosides based on some type of physico-chemical changes, as Stefano suggested it in the above comments. And that could create the negative feedback that results in CK biosynthesis.

But do you know if the glucosides that you apply can be transported to inside the cells? If so, then maybe use the glucoside transport-impaired mutants to see if there is still physiological response. If the active transport exists, then maybe that process can signal the imbalance in CK homeostasis.

Also, why are you sure the CKX genes can not degrade N-glucosides? Previous papers show there are some variants of this gene that are more specific to 9-glucosides than to CK free bases. So again, maybe it is worth to test the response of these particular CKX mutant plants on the 9-glucoside treatment. If the CKX activity takes place maybe it increases the substrate availability for de novo CK biosynthesis that you noted? So, if the extra supply of CK substrates would not be available for the mutant plants, therefore no de novo synthesis would be observed?

Overall, it is difficult to imagine that the inactive and irreversible forms of CKs are produced and stored somehow by 'unaware' plant cells until they naturally die.

Hope it helps.

Anna Kisiala I'm not sure whether it goes inside of cells. I only know it is uptaken by the plants. Actually, Petr Klíma here recently tested uptake of CKNGs by tobacco BY-2 cells and saw nothing.

I did not say that CKX does not degrade CKNGs, did I? In contrary, in the above comment I said that treatment with radioactive tZ9G showed only degradation products.

But you may be up to something. I myself were thinking that it doesn't have to be increased synthesis, but decreased degradation. But I was thinking more on the expression level.

But I do not understand what you mean with the ckx mutants? Could you elaborate more?

Hey Thomas,

Maybe i misunderstood the description in your original post. When you were questioning the option for hydrolysis of the glucosides, I assumed that CK hydrolysis is regulated by the CKX genes.

For sure decreased degradation of active CK fractions would work based on the similar mechanisms - to keep the CK homeostasis on the optimal level - I agree with that. But then, if this was the case you should not observe increased activity of CK biosynthesis genes, right?

Actually, this is exactly what the treatment of CKX deficient mutant plants could help revealing. The excess of exogenously applied 9-glucosides wouldn't be degraded but instead would accumulate in the mutant plants. If the physiological effect is a result of increased biosynthesis - you could not observe it in the mutant due to the lack of the extra substrate. If the theory about the reduced CK degradation is true - then you will need to came up with ideas for more experiments..:)

Hi Anna,

oh no. By hydrolysis I meant hydrolysis to glucose and CK free base.

Well, the decreased degradation may elevate levels of active cytokinins which in turn modulate the expression including additional CK biosynthetic genes.