Wo is wrong? The surviving plants or WE?

Let's see in the long run.....

Reto

J.D. Franco-Navarro what actually is disturbing you is;

Q: No changes in MDA (reduction in the other nutritional treatments???????) A: Yes, MDA remain unaffected among all treatments, not just treatment C. This is probably because, oxidative stress may not initiated at all possibly due to early stress acclimation response.

Q-Increase in CAT y H2O2??????? A: Within cells, H2O2 is produced during i. Photorespiration inside peroxisomes ii. By the action of SOD via conversion of superoxide anion into H2O2. iii. By apoplastic NADPH oxidases fro stress sensing, amplifications and subsequent signaling. iv. Besides, H2O2 contributes to a variety of developmental responses as well. Among various ROS produced within cells, H2O2 has a relatively longer half life that allows it to diffuse through membranes. So, increase in H2O2 in the presence of increase in CAT activity is quite possible and can be linked with stress signaling. In your case, you just only presented the results of CAT activity, but no information of SOD, POD, APX, GR, GSH and related enzymes. At least information regarding SOD-CAT-POD cycle should be determined. Such information about regulation of other enzymes is important to reach any final conclusion.

Q: a decrease in PROLINE???? A: Proline can also serve as source of nitrogen. Again for plants that were drought stressed for two weeks, proline accumulation is not always the case. A wide range of compounds serve this purpose including soluble sugars, total free amino acids and most importantly inorganic ions including K, Ca, P and Cl. Inorganic ions always serve as a very cheap osmotica to balance plant water relations. Also in the case of stomata regulation, you must know that both K and Cl are predominantly involved in stomata opening. So you did not determine sugars, K, Ca, Cl or P which I believe played a role in your case.

Q: An increase in PHENOLIC compounds is logical due to the increase in H2O2, but it has no sense in plants that are more tolerant to drought. A: Tobacco is plant from Solanaceae family rich in alkaloids and diverse secondary metabolites as indicated by shikimate pathway and some other as well. So, increased production of alkaloids including phenolics, polyphenols, and other related compounds is a natural in spite of control conditions. Yes, we can relate these with increased H2O2 but we are lacking so much data here. Above all, there is a lot of information is missing to completely understand the trend. Missing information includes; 1- How drought stress was applied (PEG or missing irrigations) 2- What is the level of drought stress? 3- This experiment was conducted in soil, sand, hydroponics etc. ?? 4- This is misleading. “Tobacco plants were subjected to 3 different nutritional treatments (A, B, C) for 3 weeks, and also were treated under two irrigation regimes during 2 weeks (Well-watered plants or Control plants, and Water deficit treated plants or drought plants)”. What I assume is first plants were grown for seven days followed by the application of drought stress alone and in combination with nutritional treatments for two weeks? 5- Also how nutritional treatments were applied? Spray, root zone or priming? 6- Determination of ABA if possible?

Answer to these questions is very important to understand what’s actually going and/ or what went wrong. My assessment -based upon your statements and statistical analyses is as follows.

Treatment C improved plant growth, water relations, water use efficiency and thereby resulted in drought tolerance among 21 day old drought-stressed tobacco plants. First of all, I suspect the stress duration of two weeks is short. During initial period, osmotic adjustments via the involvement of inorganic ions become very prominent. If plants fail to achieve tolerance, then the second step is the production of compatible solutes like proline, sugars, polyols, glycinebetaine and vice versa. And if stress is prolonged, increased ABA production leads to stomatal closure. In conclusion, stress duration was perhaps short specifically for a plant that is metabolically diverse. This could have differently affected any other glycophyte but not tobacco. Secondly, inorganic ions played a role in lowering of water potential via a decrease in osmotic potential within plant root cells. This may have contributed to better stomata regulation and water use efficiency. You can mention in the title of your manuscript “Early responses of tobacco to short term drought stress” or early stress acclimation response or anything this sort.

If you need my assistance to discuss your results further, I’ll be happy to contribute. Best Regards, Fahad

Hello J.D. Franco-Navarro

Your question can’t be answered as @Fahad Shafiq has correctly pointed out at the end of the comments because there is almost complete lack of information about the methods. The study seems to have been done with rather small plants, and in such a way that there would be a strong interaction between nutrition and water supply, making interpretation very difficult. Let us assume you grew the plants in soil, applied a liquid fertilizer and allowing some plants in each nutrient treatment to dry by stopping watering, while the control was given ample water. That is a standard molecular biology/biochemistry approach to such studies.

Which was the treatment supplying the most nutrients - A I suspect, with C supplying least. So plants in A would grow better than in C, and have more, larger leaves. When you started the drought treatment plants in A would dry the soil much faster than those in C – simply a matter of greater surface area in A. So plants in A would become severely drought stressed before those in C, which would appear to grow and be much more `drought resistant’. They would also have very different contents of metabolites. The lesson from this – if my supposition is true - is that it IS ESSENTIAL TO COMPARE PLANTS AT THE SAME RELATIVE WATER CONTENT. If this is not done then the study is invalid. I suggest my paper explains the problem and ways of doing such studies correctly – it focuses on GM plants but the problem is absolutely the same for all studies involving water supply.

Lawlor DW. Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J Exp Bot. 2013 Jan;64(1):83-108. doi: 10.1093/jxb/ers326. Epub 2012 Nov 16.

By ignoring the complexities involved with water supply in solid matrices and the effects of different rates of water loss, studies of the metabolism, genetic modifications etc are invalid. Hope this rather strong critic does not apply to your study. Please provide the necessary information about methods.

Best wishes

David

Fahad Shafiq David W. Lawlor

I apologize for the lack of data (I hide information regarding the co-authors of the experiment). I will try to explain a little better what the experiment consisted of:

Model organism: Tobacco (Nicotiana tabacum L.)

Pot size: 7.5 L pots (pot size 20 cm × 17 cm × 25 cm)

Substrate: a mix of perlite:vermiculite (4:6)

Treatments: Seeds were sown and two weeks later (15 days after sowing, DAS), seedlings were transplanted to 7.5 L pots. Then, plants were subjected to three different nutritional treatments. After 30 days (45 DAS), in addition to the three nutritional treatments, plants were also subjected to two irrigation treatments: optimal irrigation (control; CTR), in which pots containing tobacco plants were irrigated up to 100 % field capacity (3.5 mL g-1 substrate) throughout the experiment, and moderate sustained water deficit (WD), with pots irrigated every two days up to 60 % of field capacity (2.1 mL g-1 substrate) for 20 days (64-65 DAS).

Hello J.D.!

Thanks for the information. The experiment is as I suspected in my response to your question. So the answer is that, unfortunately, the water-stress analysis is incorrect. The way to do such experiments is given in the paper I referred to. It is essential to measure the water-status (I consider relative water content - RWC- is the best indicator) of the plant. By attempting to apply a proportion of `pot capacity' the study gets even more complex to interpret as the plant's RWC will vary depending on the timing of water application. So your inexplicable data can be explained but not in a way that you would have wished. Sorry. The problem you have become engaged in is a major one in plant biology, and stems from the failure of molecular biologists and biochemists to really make efforts to understand plant water relations and particularly the characteristics of soils. As an old-fashioned physiologist/ecologist I would think this, wouldn't I?

Very best wishes

David

David W. Lawlor explained the scenario pretty comprehensively. The way drought stress and nutritional treatments were applied seems inappropriate which caused unexpected variations in your case. Movement of water through soil-root interface is governed primarily by differences in water potential and a combination of root pressure, and transpiration pull (also connected with overall plant water status). So by adding different nutrient doses via root zone, your J.D. Franco-Navarro treatments actually reduced water potential (More in A and least in C) and thereby affected subsequent plant responses.

Best regards

Fahad

Excellent discussion (above). Even an old Biologist (Bioenergetics) can learn from engaged experimentators. Thanks and best regards.

Reto J. Strasser, ([email protected])

Very informative discussion which is useful for everybody interested on drought studies. Thank you Dr. Lawlor and Dr Shafiq for the comprehensive comments.

I absolutely agree with Reto and Thusari. What I pretended is to start a great discussion, and Dr. Shafiq and Dr. Lawlor did it. Thanks.

Hello FD,

Thanks for sending the papers - I have had problems down-loading all of them (4?) and wonder if you could send them again. I read two (J Expt Bot and Plant Cell?) and see that the effect of salinity is to increase succulence and alter a number of physiological characteristics, as in sugar beet which I worked with very long ago. Going back to your original data which were unexpected: what were the"nutritional treatments" A, B and C? I seem to have not understood - might be important.

Regards

David

Very interesting and informative discussion. I am also working on plant metabolomics, this discussion is really fruitful.

Thanks to all experts.

Dear @ J.D. Franco-Navarro ,

Find some similar researches in the article below:

Article Comparative analysis of overexpressed Fragaria vesca S-adeno...

Dear Ákos Mendel thank you for your comment. I have just sent your paper to my lab group in order to solve our question. Thank you very much indeed.

Dear J.D. Franco-Navarro ,

I hope I could help You! Best wishes!