A noncanonical auxin-sensing mechanism is required for organ morphogenesis in Arabidopsis
Authors: Joyita Deb
Dolf Weijers
Laila Moubayidin
Lars Østergaard
Jiří Friml
Karim Sorefan
Pauline Stephenson
Manoj Valluru
Alejandra Freire-Rios
Sara Simonini
Journal: Genes & Development (2016)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Arabidopsis; ETTIN; IAA; auxin signaling; plant development
Affiliations: Department of Crop Genetics, John Innes Centre, Norwich NR4 7UH, United Kingdom., Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, the Netherlands., Institute of Science and Technology (IST) Austria, 3400 Klosterneuburg, Austria.

Abstract:  Tissue patterning in multicellular organisms is the output of precise spatio-temporal regulation of gene expression coupled with changes in hormone dynamics. In plants, the hormone auxin regulates growth and development at every stage of a plant's life cycle. Auxin signaling occurs through binding of the auxin molecule to a TIR1/AFB F-box ubiquitin ligase, allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently ubiquitinated and degraded via the 26S proteasome, leading to derepression of auxin response factors (ARFs). How auxin is able to elicit such a diverse range of developmental responses through a single signaling module has not yet been resolved. Here we present an alternative auxin-sensing mechanism in which the ARF ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. This noncanonical hormone-sensing mechanism exhibits strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) and is important for coordinating growth and patterning in diverse developmental contexts such as gynoecium morphogenesis, lateral root emergence, ovule development, and primary branch formation. Disrupting this IAA-sensing ability induces morphological aberrations with consequences for plant fitness. Therefore, our findings introduce a novel transcription factor-based mechanism of hormone perception in plants. 

Retracted: No

PubPeer comments:
 ![file] Figure 4. ETT and IND protein interact in an IAA-sensitive manner. (Au2013D) BiFC with pYFPN-ETT and pYFPC-IND in the presence of IAA (B), NAA (C), and 2,4-D (D). All hormonal treatments were with 1 mM in lanolin. (E) Fluorescence quantification (CTCF) of split YFP signal between ETT and IND without treatment and with IAA, NAA, and 2,4-D. (*) P lt; 0.01; (***) P lt; 0.0001. Error bars show the standard deviation. (F) Y2H assays with increasing concentration of IAA, NAA, and 2,4D. Bars: Au2013D, 50 u00b5m. **Panels A and D seem to originate from the same picture.** ![file] , 
 Hi Xestia Homogena, thank you very much for spotting this error. We are now working to replace the duplicated image with the original one coming from the same experiment dataset. Best, Sara , 
 Dear Pubpeer Xestia Homogena Thank you very much for identifying this error. We have gone back to the original images and have verified that the image used in both 4A and 4D is indeed from the untreated control experiment. We have identified the images from the 2,4-D treatment (used for the quantification in 4E) and will build a revised figure for publication as an Erratum in Genes Dev. We are very grateful to you for having notified us so that this error can be corrected. All the best Lars Ostergaard , 
 Dear Authors. How could this happen? How do you explain the fact that the image was not only duplicated, but also tilted, stretched in one direction and its brightness was changed? , 
 I wonder how people respond when they get such questions at conferences or invited talks. Should have probably happened somewhere. , 
 Dear Simulium Chromatinum and Pubpeer Thank you very much for your interest in these data. It is true that the three nuclei you picked out are imaged twice. This is not an error. These nuclei were imaged independently with two different objectives (10x and 20x) and although the settings were the same, the 20x gives a better resolution than the 10x; hence the different values. The values are: 1 - 7 -- gt; 20453.5209 vs 20793.46855 2 - 8 -- gt; 25498.58933 vs 19878.02362 5 - 9 -- gt; 14955.00461 vs 12556.80181 The average of the 20 values for the Mock treatment listed in the Supplemental Figure for the Corrigendum is 20736.15238 u00c2u00b16541.048891 (stdev). Repeating this calculation by eliminating 1-2-5: 20803.055 u00c2u00b16879.1482 (stdev) Repeating this calculation by eliminating 7-8-9: 21253.6915u00c2u00a0u00c2u00b16806.417 (stdev) Repeating this calculation by eliminating 1-2-5-7-8-9: 21458.8315u00c2u00a0u00c2u00b17244.5514 (stdev) The p-value associated with these sets isu00c2u00a00.989227 which means that all these analyses give the sameu00c2u00a0results. Regarding the other issues, we are not quite sure what is being suggested, but happy to respond. - Firstly, we did not do a careful analysis of the stomatal closure in response to the treatment of auxins. I wonder if Simulium Chromatinum is referring to a paper by Tanaka et al. (2006) in J. Exp. Botany, which shows that auxin (NAA) inhibits ABA-induced stomatal closure. In order for the authors to obtain this effect, they treated with NAA and ABA in combination, so a very different assay from the one that is carried out here. - Secondly, regarding inhibition by 2,4-D on yeast growth, we did not observe this effect with the concentration used here. In fact, as opposed to IAA, 2,4-D (and NAA) had no effect on the ETT-IND interaction in yeast. - Finally, the reason that no residual lanolin is visible is because the infiltration with lanolin paste is done on the upper surface of the leaf, while images are taken of the lower side. This is advantageous both because of difficulty in taking a decent picture of a leaf when lanolin has been applied, but importantly also because we are then not measuring the effect in cells that have been in direct contact, but rather in cells that have taken up the auxin through diffusion. As stated in the Methods section  Exogenous IAA, NAA, and 2,4D applications were performed by mixing lanolin with the hormone as described previously (Reinhardt et al. 2003); a thin layer of lanolinu00e2u0080u0093hormone mixture was applied on the infiltrated area 24 and 48 h after infiltration . I very much hope this will clarify the issues raised. Best wishes Lars Ostergaard , 
 Thanks for providing the original data. In your answer, you write: u201cIt is true that the three nuclei you picked out are imaged twice. This is not an error. These nuclei were imaged independently with two different objectives (10x and 20x) and although the settings were the same, the 20x gives a better resolution than the 10x; hence the different values.u201d I see a few major problems with it: 1. You can not measure two times the same object and use it for statistical analysis. This **is** an obvious **error**. 2. Utilizing two different objectives represents a typical example of **NOT using the same settings**, which is unacceptable for signal quantification. Since scale bars are missing from the images, how can we know which objective was used for each image? It looks like you have used different magnifications for mock treatment (varying sizes of the nuclei between images), only small magnifications for 2,4D, and again various magnifications for NAA. **Data acquired with different settings (including objectives) canu2019t be compared.** 3. In the images provided one can see that some nuclei were omitted in the quantification process. In the case of IAA treatment, one can see two very bright nuclei in the middle of the top left panel. Why were these not measured? There are **only 10 nuclei ** quantified in this case, so at least 20% of the data was left out for no reason. In my opinion, these serious issues require repetition of the experiments and a correction of the paper. , 
 Dear Xestia Homogena I am delighted with the interest there is in this paper and in particularly this experiment. We very much acknowledge your views and appreciate your motivation, but I am afraid we do not agree on this occasion. Although we donu2019t agree with you that one cannot use the same object for the analysis in the way we have done, you can tell from our previous response, that even removing these data points would not change the conclusion. On your last point, yes there are two signals that were abnormally high in the IAA experiment. Since these were the only strong ones we observed, we took the decision to omit them as clear outliers from all the additional data points. However, we have taken a completely transparent approach to make clear for everyone to see and form their own opinion exactly as you have done. So, Iu2019m afraid we will have to consider this a case of having to agree to disagree. All co-authors of the paper have been consulted on this response. With best wishes Lars Ostergaard , 
 There are even more duplications in this original data set. Please have a look at the top panel for 2,4D treatment (low magnifications): http: genesdev.cshlp.org content suppl 2017 10 05 30.20.2286.DC2 Supplemental_Figure_4_raw_data.pdf ![file] All is the same specimen images three times. Again, note the very different fluorescence intensities between identical locations within three images... , 
 http: genesdev.cshlp.org content 31 17 1821.full image corrected but still faulty! Same cells imaged in Additional Supplemental Material: https: imgur.com a QvdyD Although the same nuclei (1 and 7; 2 and 8; 5 and 9) fluorescence values are different, factor 0.8 - 1.3 Other issues with Fig. 4: -all stomata closed in presence of auxins -high concentrations of 2,4-D inhibit yeast growth -no residual lanolin visible , 
 There is another image duplication in the data underlying Figure 4a https: imgur.com a fqc2Z Specimen and objective slightly moved. What is supposed to be an experimental replicate is in fact a duplicate. Note the very different fluorescence intensities between identical locations within the two images. This shows how inappropriate the method is for quantitative purposes. The main conclusion of the manuscript - a non-canonical auxin sensing mechanism at work - is solely based on data presented in Figure 4 (IAA preference). Surprisingly, mutants of the classical auxin receptor have not been tested to support this claim. , 
 Two images in supplemental figure 3D appear to overlap. This doesn t seem to be discussed in the prior posts. Could the authors please check? *** ![file] , 
 1 u2013 Figure 3 panel A. Same image as in raw material u201cMock treatmentu201d. The image has been flipped and tilted. 2 u2013 Figure 4: -tPanel A. Same image as in raw material u201cMock treatmentu201d. The image has been flipped and tilted. -tPanel B. Same image as in raw material u201cIAA treatmentu201d. The image has been flipped and tilted. -tPanel C. Same image as in raw material u201cNAA treatmentu201d. The image has been flipped and tilted. -tPanel D: Same image as in raw material u201c2,4D treatmentu201d. The image has been flipped and tilted. 3 u2013 About raw material the authors reported: u201c*Moreover, we have added a raw data file as an Additional Supplemental Material link associated with the original article with **the raw images of three independent experiments** that were used for the quantification in Figure 4E (Figure_4_raw_ data.pdf). **For each condition, we have included a picture taken with a low magnification (a 10u00d7 objective). In addition, we have included four more images at higher magnification (a 20u00d7 objective)** and have repeated the intensity quantification using the nuclei that are labeled with a number*u201d. -tDespite the three independent experiments claimed by the authors, Mock and 2,4D treatments clearly show duplicate images (sometimes with different color intensities) indicating one single experiment. -tTwo of the four images at 20X in Mock treatment are duplicated and the samples 1,2 and 5 are the same as the numbers 7,8 and 9. Nevertheless, these data were used for statistical analysis. 4 u2013 Figure Supplementary 3D: image duplication between mock and 2,4D treatment. ![file] ![file] ![file] ![file] ![file] ,