Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture
Authors: Yangrong Cao
Nagib Ahsan
Hong Li
Daewon Kim
Jay Thelen
Gary Stacey
Dongqin Chen
Journal: Nature Communications (2017)
Discipline: Multidisciplinary
Keywords: ATP; stomata; gas exchange; photosynthesis; drought tolerance
Affiliations: Division of Plant Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, 65211, USA., State Key Lab of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China., Division of Biochemistry, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, 65211, USA., Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital,, Providence, RI, 02903, USA., Division of Plant Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, 65211, USA. ., Division of Biochemistry, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, 65211, USA. .

Abstract:  In addition to acting as a cellular energy source, ATP can also act as a damage-associated molecular pattern in both animals and plants. Stomata are leaf pores that control gas exchange and, therefore, impact critical functions such as photosynthesis, drought tolerance, and also are the preferred entry point for pathogens. Here we show the addition of ATP leads to the rapid closure of leaf stomata and enhanced resistance to the bacterial pathogen Psuedomonas syringae. This response is mediated by ATP recognition by the receptor DORN1, followed by direct phosphorylation of the NADPH oxidase RBOHD, resulting in elevated production of reactive oxygen species and stomatal closure. Mutation of DORN1 phosphorylation sites on RBOHD eliminates the ability of ATP to induce stomatal closure. The data implicate purinergic signaling via DORN1 in the control of stomatal aperture with important implications for the control of plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield. 

Retracted: No

PubPeer comments:
 Fig6a,b: How did they successful detect luminescence signal by a normal fluorescence setting? Perhaps they have a trick not written in the text? or they mistakenly perceive the false signal as a luminescence result? **Fig6a** ![file] **Methods** ![file] , 
 Yes...the figure legend is incorrect since we clearly were measuring luminescence, not flourescence. Interesting how such mistakes can escape detection considering how many times the manuscript is read and by various different people. However, the rest is correct in that we used the CCD camera for light detection, which is shown. , 
 Possible band duplication and blot reuse in figs 2 and 3 ![file] ![file] Can the authors supply original blots? , 
 We were notified concerning the questions raised by Trigonopterus Ptolycoides (TP) via anonymous letter that was not sent to me but to my Department chairman and also to our Office of Research, which seemed odd to me if the intention was to seek an explanation for what was claimed to be irregularities in two figures in our paper. This letter arrived while I was traveling and, hence, when I returned I consulted with the first author of the paper and, as described in detail below, we provide a strong rebuttal to the claims made, as well as providing the original blots as requested. Addressing the figures in the order they appeared in the paper: Figure 2b. The claim was that we cut and pasted three gel blots lanes to give the appearance of conducting three, separate experiments. First, the triplets in the various lanes do not, as claimed by TP, appear similar to us at all, even if one manipulated the contrast. This is also clear from the original blots that are shown below. Note that only the center lanes from the original blot (of Fig. 2b, left figure below) were used in the paper. Areas of the gels used in the published Fig. 2b are boxed in red. ![file] Figure 3a. The claim here is that we manipulated one figure so as to make it appear that two, independent experiments had been conducted. The basic problem here, we believe, is that TP did not fully understand how such experiments are performed. This figure shows in vitro co-IP. This experiment is done by mixing the two, epitope tagged proteins, along with the GST-beads. An aliquot is then taken of this to run the input gel (shown on the right above) and then pull-down is done which generates the actual IP gel that is shown on the left above. I mention this specifically because the shadows in the gel that seemed to be the primary concern of TP are the result of the fact that the same protein solutions are used to generate both gels. Note that both gels are visualized using anti-GST antibody so it is not at all surprising and, indeed, expected that they should be near identical. However, beyond that, as can be seen above, two separate gels were generated to create the Fig. 3 shown in the paper. To make this even clearer, we put red circles around specific areas of the two gels that are obviously different. We actually did not include the input gel picture (Fig. 3, right figure above) in the original submission, given that they would appear identical. However, one reviewer insisted on including this and so we did so in the final version. I would also draw the readeru2019s attention to Figure 4b and 4f in the paper by Li et al. 2016. Nature Commun. DOI: 10.1038 ncomms14356. Fig. 4b gives a very good example of where a similar Co-IP experiment was performed and the GST input gel was included (similar to our paper). Note that, again similar to our Fig. 3a, the anti-GST input gel is identical that of the anti-GST IP gel. Fig 4f in this paper is an example where the anti-GST input gel was simply omitted. Another published example is Figure 1a in the paper by Jeong et al. 2019 https: doi.org 10.1038 s41467-019-09086-0 where again the input gel is shown and appears identical that of the IP gel. Indeed, this would be a good one for TP to claim was the result of manipulation of the exposure. Finally, the figures that were questioned in the paper represent data that were replicated using independent means by other experiments that were presented. Hence, the conclusions of the paper would not be affected even if there were irregularities. However, clearly this was not the case. I have no complaints with TP wanted to maintain his her anonymity. However, I do think it is simple courtesy to first bring such questions to the attention of the authors and allow them to respond. Clearly, if they do not respond or if their response is not satisfactory, then taking this higher within the administrative chain would be acceptable. This was not the procedure that TP followed. Regardless, it would be good for pubpeer to institute a procedure where authors would be automatically notified if comments or concerns were raised about their published work. Submitted by Dongqin Chen and Gary Stacey, March 29, 2019 , 
 Thank you for your responses but I do not feel that they fully address my original points. Your responses do not show the edges of membrane or films that could be used to rule out the points raised. A larger crop is still a crop. In particular for fig 3a, where I fully understand the methodology and experiment, the background artifacts ringed by the red polygon are the same between the 2 images but visible to different degrees (i.e. suggesting different exposures of the same blot). The artifacts highlighted in your response are consistent with different exposures (i.e. handling damage to file, processing effects, dust specs. etc). How can you account for the artifactual similarities (particularly those that cross lanes and have not  run in a manner that would suggest that they correspond to proteins)? In response to your comments about alerts I clicked the button to notify both first and last author using the info that PubPeer have when I submitted my comments. I apologise if that didn t make it to you. I did not notify your institution. , 
 #5 We used the BioSpectrum imaging system to capture the WB result. We did not size the blots identically in every image. There was no selective cropping of the images in our previous rebuttal statement. The point you make is based on your assumption that the u2018ghostsu2019 that you see in the gel are due to unique gel imperfections and, hence, should not appear in images taken from two, separate gels. We understand the point but question the assumption. We do not have an explanation of what the u2018ghostsu2019 are caused by but, since they do indeed appear in images taken of two, separate gels, we have to conclude that they are caused by something in the extract and, hence, appear in both the input and Co-IP gels (see previous rebuttal). To make this point even clearer, for Fig. 3a, we adjusted the images so that exposures were near identical (see below). Following along your logic, if these were indeed images of the same gel then imperfections, unique shapes of protein bands, etc. that appear in one image should be identical in the second image. This is clearly not the case. The red arrows we added show several, clear differences between the two blots in the u201crunu201d lanes, which clearly cannot be attributed to dust, etc. In a word, the two gels, compared at the same exposure are clearly different, which cannot be attributed to cropping or some other manipulation. Again, although we are happy to respond to any criticism, the net effect is that none of this has any real impact on the overall conclusions of the paper. ![file] , 
 #6 One of the major issues that TP has with Figure 3 above, is the shadow that appears on both gels (see red outlined area on his original post). As we indicated, we have no ready explanation for this and do understand how he suggests that this reflects the use of one gel for both images. However, we feel that we have now shown conclusively that two gels were used (see our rebuttal above). However, the puzzle over these shadowed areas remains. Again, we have no concrete explanation but perhaps it is pertinent that the two gels were electroblotted at the same time using our dual apparatus. Hence, if there was some impurity or other issue that arose through the electroblotting process, that could be an explanation. , 
 I agree with TP. I ve run enough gels and HRP based westerns in my life to know that subsequent exposures of the same blot can have slightly different shaped bands or specks on. I also know that when you get weird shaped blobs on blots in the exact same place as TP ringed in red the images came from the same blot. You can see them much more clearly on your image above. If they were the same shape but in a different position then they could be from the imager where the blot was sitting. But they aren t, they are in the exact same place. They also don t look like something that has run on the gel. Do you really think that 2 separate gels would produce such similar curves, blobs and smears than span across multiple lanes? And your point about not affecting the overall impact or conclusions? How are we meant to trust ANY of the data in this paper if some of it looks potentially suspicious and the rebuttals are less than convincing, particularly in light of: https: pubpeer.com publications F8F04CD5607070BEF02BB81BD0C152] and https: pubpeer.com publications B8298E6D84844FADC363F1A78A23E4] and https: pubpeer.com publications 2AE11A10458E12AEAD855BB113F48A , 
 #7 A few comments: (a) In comment #4, features outlined by the two left-most red ovals and the one right-most red oval were claimed as evidence that the two images derive from  two separate gels . This is surprising, given that the overexposed versions in #6 indicate that these features are beyond the gel boundaries (i.e., the rectangles with darkest background). (b) I don t agree that you have  shown conclusively that two gels were used . I agree the images do not seem identical, but they could, for example, represent two different exposures or different image acquisitions of a single blot, unless some  fingerprint feature of the gel distinguished them. That s why the similar splotches are critical. (c) The splotches suggest some sort of background signal caused by a contaminant, uneven blocking, membrane handling pressure, etc. It certainly is conceivable that there could be an impurity in the electroblotting transfer apparatus (as raised in #7), which was thereby transferred to two separate blots. If so, however, they should have **equal dimensions**. But they do not. In the  input blot image, both the protein band positions and the splotches are elongated vertically compared to the pull-down blot image. So, as presented, the input gel appears to have been run farther, and the impurities from the electroblotter also just happened to become stretched out proportionately. Alternatively, the gels were run the same distance, and the original splotch patterns had equal proportions, and one of the images was later stretched or shrunken vertically compared to the other. So, are you certain that these  original images have not been re-sized? Or perhaps has the vertical dimension of one been altered compared to the other? (d) The  original images are quite poor resolution (much poorer than the published versions) and obviously have been subjected to JPEG image compression, as compression artifacts are clearly evident (e.g., transfer them into Photoshop and enlarge). The concerns here would be clarified far better by posting true originals -- i.e., raw images that have not been re-sized, down-sampled, or compressed. The ones posted here are of such poor quality that they actually obscure the main issues raised. , 
 We are continuing to look into the issues raised and will post the results of our inquiries when available , 
 The issues raised in this posting, as well as a string of rather nasty tweets, were investigated by an independent review committee composed of three senior faculty organized by our office of research. They investigated these figures, as well as all of the data presented in this paper, looking at all of issue of malfeasance and whether the underlying scientific conclusions were supportable. The final conclusions of this committee conveyed to me and the first author of this paper was that there was no evidence of any data manipulation or other issues that could be characterized as academic misconduct. Evidence for my statement can be found in the postings of Mr. Leonid Schneider who raised these issues quite aggressively and persistently with my superiors at the University. Although I understand, again based on past experience, that this will not silence the trolls that like to perpetuate such negativity, from our point of view, we consider the case closed. ,