Probably the amount of silver used in your experiment could lead that undesired behaviour, since silver its toxic by itself. So I recommend you to work better with mutants in ethylene production if they are available. You might use Aminoethoxyvinylglycine that inhibits ethylene production instead of silver.

Regards,

Thanks, Aarón; I have to ruled out this hypothesis because I had controls... Silver was not toxic in non stressed treated plants.

THIS  PROBABLY DEPEND ON THE PHYSIOLOGICAL PHENOMENON IN QUESTION. WE HAVE FOUND FROM OUR WORK ON DATE PALM FRUIT RIPENING,WHICH IS A CLIMACTERIC FRUIT, ABA TRIGGERS ETHYLENE PRODUCTION AND FRUIT RIPENING IN THE DATE .EARLIER ,ZHARG ET AL HAVE FOUND THAT ABA TRIGGER ETHYLENE  AND HENCE TOMATO FRUIT RIPENING,J.Exp.Bot,60,1579-1588,2009. ,

Yes, Muayed. Ethylene is fully involved also in fruit ripening, known from long time. It is an antagonist of ABA in osmotic stress, according to literature. However, this may not be a general rule. So, I hope someone in here may have different experiences on this regard. Thank you for you opinion.

Thank  you for your comment , as you said, ethylene is long known as the ripening hormone,but the new thing is that ABA triggers the production of ethylene in a climacteric fruit,the date palm, where ABA should have no role at all.   In  non- climacteric fruit, such as grape,ABA  is not detectable during early stages  of fruit  development, but the levels of ABA  increased to peak value as the berries entered the ripening phase,where as the reverse was true for ethylene levels. so in most of  non- climacteric fruits,ABA IS THE RIPENING HORMONE.  unlike animal hormones, plant hormones have many physiological effects, some cases are antagonistic and others are synergistic depending on plant tissue, stage of development, the physiological process under investigation, and so on, and because plant hormones do not show  a clear cut  specific effect, as it is the case in animal hormones, we speak of plant growth  substances.

In our experiment both ABA and ethylene level increased under some abiotic stress conditions and we also see stomatal closure after these increases in both phytohormone level. Since in the previous literature it was mentioned that increased ethylene level can prevent ABA-induced stomatal closure, to investigate the exact role of ethylene we used 1-MCP to prevent ethylene action and also ACC on the plants but we did not prevent stomatal closure in the conditions when both endogenious level of ABA and ethylene is high. Therefore, it seems that the antagonist action of ethylene on ABA is not a general rule.

Hi, Sasan;

This is similar to what I got with my coffee seedlings and I am trying to find out or explain. "Inhibiting ethylene" with silver thiosulphate made them more susceptible to stress than controls...  I don't have a clue for this at this moment. Does someone else have it?

Please have a look on the following paragraph which selected from our review paper (attached link):

http://m.jxb.oxfordjournals.org/content/64/12/3551.full

Sorry here is the selected paragraph:

It has been reported that cytokinins and auxins influence stomatal movements via ethylene. These plant hormones can promote stomatal opening via the antagonistic effects of ethylene on ABA-induced stomatal closure, probably through the modulation of ethylene biosynthesis (Tanaka et al., 2006). However, there are contradictory reports regarding stomatal responses to ethylene. It seems that stomata close in response to ethylene in the absence of ABA and open in the presence of ABA (Desikan et al., 2006; Tanaka et al., 2006). It has been shown that ethylene or ACC (1-aminocyclopropane-1-carboxylic acid; the precursor of ethylene) can prevent ABA-induced stomatal closure (Tanaka et al., 2005). Under drought conditions, application of ethylene increases stomatal aperture of wild-typeArabidopsis (Tanaka et al., 2005). Ethylene seems to act in the later steps in ABA-induced stomatal closure but not in the early steps (Tanaka et al., 2005). The interaction between AtERF7 (which is a member of the ethylene-responsive binding factors) and PKS3, a Ser/Thr protein kinase which interact with ABI2 and to some extent ABI1 (Guo et al., 2002), reduces the sensitivity of guard cells to ABA and induces water loss through transpiration (Song et al., 2005). It seems that ethylene acts via an ABA-independent pathway that leads to stomatal closure. The ethylene receptor ETR1 mediates H2O2 signalling in guard cells and may be a sensor for H2O2 perception in guard cells (Desikan et al., 2005). So ETR1 may be the site of ethylene and H2O2 cross-talk leading to stomatal closure (Desikan et al., 2005). Ethylene antagonizes the ABA-induced stomatal closing response after long-term O3 application in wild-typeArabidopsis plants. Exposure of plants for a long time to elevated O3concentrations (70 ppb) leads to stomatal malfunctioning in response to ABA and water stress (Wilkinson and Davies, 2009). In this case, the malfunctioning of the stomata coincided with an induction of ethylene production. On the other hand, when the plants were exposed for a short time to O3, after 6h the level of ACC and ethylene decreased to the control level; at the same time, stomata start to close (Overmyer et al., 2008). Consistent with this hypothesis, it has been shown that pre-treatment of ozone-treated plants by 1-MCP (1-methylcyclopropene; a blocker of ethylene receptors) restores the closing response of stomata to ABA or water stress (Wilkinson and Davies, 2009). 

Thanks for your reference, Sasan;

Because of my limited resources and education (plant geneticist and breeder with minor plant physiology and biomol), I choose to look this quest at the phenotypic level, less to the genotypic one (better say molecular, biochemistry). So, I try to understand my results in the light of the current knowledge. I further recognize I cannot be fully updated in a field that is quite broad an not from my background exactly. Questions like how ABA works at fine levels, which receptors, how it gets inhibited, how it interacts with other compounds (ethylene, NO, K, aquaporins, etc.) remains somewhat elusive for me, despite my efforts.

You can get very recent publications at biochemistry and molecular biology if searching google as https://www.google.com.br/?gws_rd=ssl#q=aba+receptors+in+plants

But they did not clarify my questions yet, unfortunately. Perhaps someone else could read them altogether better than me...

Hello sir.

Did you measure ethylene levels to determine if ethylene was indeed inhibited?  How did you determine that ethylene was inhibited and ABA increased?  I appreciate you opening this up for discussion.  Jake Sanchez

Hi, Jake; perhaps I was not clear enough in my previous comments. I did not measure ethylene but observed the plant phenotypic response. I was supposing if it would have an antagonic effect to ABA, then his inhibition would favor ABA action: plants would fare better than controls on osmotic stress treatment - the reverse occurred.

Dear all

We measured both phytohormones (ABA and ethylene). High level of these two phytohormones detected together with strong inhibition of stomatal opening.. I hope I help with discussion by this finding.

let me know the methodology of phytohormone estimation form leaf tissues 

For ABA measurement you can have a look on the following link.

For ethylen measurements gas chromatography was used for the air collected from growth chamber and closed containers around plants

http://m.jxb.oxfordjournals.org/content/65/22/6529.full

Sasan, how would you measure stomatal aperture from over 60 seedlings every 2 hours for 48 hours, say? This is the time frame where my results happens... So, I rely on visual clues, that are highly evident.

Luis, what was the osmotic stress level imposed? Depending on the stress level, the concentrations of ABA and ethylene differs and as well as their interaction. At mild osmotic stress, mild concentrations of ABA and ethylene may synergistic, at moderate level, they may be antagonistic and at higher levels both are detrimental to plants growth.

In your case, you have used silver ions to block ethylene. Silver ions are known to be ethylene perception inhibitors but not ethylene biosynthesis. This means, your plants still have ethylene but may have ethylene perception and signaling blocked. This ethylene may antagonize ABA, promoting stomatal opening and plants may loose more water and become more susceptible to osmotic stress. If you use any other inhibitors of ethylene biosynthesis or ethylene deficient mutants, you will see different response of plants to osmotic stress. At the end, the balance of ABA and ethylene is important that may control the stomatal more sensibly than either of the hormone alone. Please check your results in this direction. Hope this helps. 

 Thanks, Ravi; my purpose is to understand what is happening with my coffee seedlings when they are subjected to water osmotic stress. Remember coffee is a perennial tree, quite difficult to study stress in field conditions because of the high plant size. I would say my system is severe: it kills my “wild” or “normal” seedlings in 24-48 hours. I established a standard test after experimenting several doses and time with PEG 6000. The dose I chose kills my seedlings, so, it is convenient to select mutants or variants if the eventually comes up. Some results appeared quite interesting and I seek understanding them, as you gave some hints on ABA & ethylene. Yes, silver blocks ethylene from binding to its receptor, that has a cooper ion: silver displaces it, I read. So, there should be free ethylene molecules within the cell environment.

The reason I started using silver had nothing to do with ethylene, but to block aquaporin gating. Silver is a potent “in vitro” aquaporin gating blocker. If achieving this, cytosol would stop loosing water out and the plant would fare better. As we understand now, the reverse happened when moving experiments from “in vitro” to “in vivo”. An additional information: I started using Hg at the beginning. Because of the risks of Hg, I moved to Ag. Hg is very dangerous to manipulate, as we know.

Now, you say this “free” ethylene “may antagonize ABA, promoting stomatal opening and plants may lose more water and become more susceptible to osmotic stress”. This is very interesting and would provide an explanation to my results. So, I ask if you please could provide evidence (literature?) for it.

Regarding to mutants, I don’t have them for coffee; also I would be happy in trying other kinds of inhibitors if I had them.

Now it is getting interest when you mention that you used silver for blocking aquaporins. Remember, aquaporins are not just present in the cytosol but also in other tissues and compartments. How did you apply silver? is it root feeding or foliar spray. It makes sense that when you apply silver to block aquaporins, silver can block aquaporins somewhere else too. ABA is known to regulate plant hydraulics via aquaporins. If aquaporin is blocked in the root, which means high root hydraulic conductance and impaired root water uptake and no root growth. This means, plants will become highly sensitive to osmotic stress. So, the silver you applied has two-fold effects: 1. via influencing ethylene; 2. via plant hydraulics. It is most likely that both pathways may operate in your plants.

We used an efficient technique based on the fluoresence of chlorophyll under non_photorespiratory conditions to measure the stomatal responsiveness to ABA. For Arabidopsis we developed a nice method in a way that stomatal responsivness of 32 leaf samples can be measured in one time. If you are interested you can have a look on our publication. 

Hi Ravi

This statement "At mild osmotic stress, mild concentrations of ABA and ethylene may synergistic, at moderate level, they may be antagonistic and at higher levels both are detrimental to plants growth" looks really interesting

Do you have some supporting indication for it?

Thanks, Ravi;

Just making a short comment by now, I tried silver in both ways (root solution and leaf spray) and got same results...

Hi Sasan, there is no single paper showing these relations between aba and ethylene. However, if you go through literature, you do find such relations between aba and ethylene.

Hi Luis, the same results means you have both possibility in the your results. Analyst your results in both ways. There are some interesting concepts of hormones and hydraulics and a lot needs to be studied. Good luck.

I just like to share this recent PNAS paper related to this discussion - ABA., NO and water stress.

http://www.pnas.org/content/112/2/613.full

Hi, guys;

I think I found an interesting paper with a good answer: Ethylene mediates stomatal closure, so, silver blocks the ethylene action. Thanks for all your comments.

http://jxb.oxfordjournals.org/content/62/8/2657.short