Nobody Expects the Spanish Inquisition!

Pedro Rodriguez is professor of plant biochemistry at the Instituto De Biología Molecular Y Celular De Plantas (IBMCP) in Valencia, Spain, where he works since 1998. Aneurus Inconstans is an anonymous image integrity expert who specialises in plant sciences. The two had a lively debate on PubPeer, others joined and now 15 of Rodriguez papers are flagged for image duplications. And then I had an even livelier email exchange with Rodriguez, which somehow included him requesting a retraction of one of his problematic papers, and then calling me a “Spanish Inquisition”.

The journals affected in this affair are all issued by learned societies, namely the American Society of Plant Biologist (ASPB) and the Society for Experimental Biology (SEB), and count as the most respectable in the field of plant sciences. These journals also have generally a good record of cracking down on data manipulation. Even if plant sciences are not free of fraud (see for example Olivier Voinnet affair), the field is much less fraud-tolerant, fake plant science gets retracted.

Did Plant Cell learn from Voinnet Affair?

One of the journals discussed here, The Plant Cell, even dismissed their just-appointed Editor-in-Chief, Sally Assmann, because of research malpractice in her lab:

Sally Assmann, right EiC choice for Plant Cell?

Unlike in biomedicine, where even the most notorious research fraudsters constantly get rewarded to drive home a point, and even nobodies with their fake papermill products are safe from retractions most of the times. This is why I prefer writing about plant sciences rather than about toxic dumps called cancer, ageing or regenerative medicine research. But of course it’s not black-and-white, there are very honest biomedical journals like Journal of Biological Chemistry or (since recently) PLOS One, and very disappointing exceptions among plant science journals, the commercial ones for sure, but also the society-run Journal of Experimental Botany here.

Meaningless and pseudoscientific potatoes

“We want to make a retraction”

So, without any further ado, here is the paper Rodriguez decided to retract:

Julia Santiago, Americo Rodrigues, Angela Saez, Silvia Rubio, Regina Antoni, Florine Dupeux, Sang-Youl Park, José Antonio Márquez, Sean R. Cutler, Pedro L. Rodriguez Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs The Plant journal (2009) doi: 10.1111/j.1365-313x.2009.03981.x

Elisabeth Bik joined Aneurus Inconstans in the treasure hunt, and found more:

Penultimate author Sean Cutler, professor at University of California Riverside commented:

“Thank you for drawing attention to these issues. The figures/data in question did not originate from my laboratory at UC-Riverside and I do not have copies of underlying raw data used for the figure preparation nor do I have a simple explanation for the irregularities noted. I have contacted the corresponding author of the manuscript and he has indicated that he will respond.“

On 24 June 2021, Rodriguez informed the journal (with me in cc):

Dear Editor,

We want to make a retraction corresponding to the article:

Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Park, S-Y., Dupeux, F., Marquez, J.A., Cutler, S.R. and Rodriguez, P.L. (2009). Modulation of drought resistance by the abscisic acid-receptor PYL5 through inhibition of clade A PP2Cs. Plant J 60, 575-588.

Recently we realized that some images were used wrongly in the paper, so I want to retract this article.

The key message of the paper is very solid and results have been reproduced independently in many laboratories, but I find unacceptable the wrong use of some images during figure preparation

Please, let me know the procedure

Pedro Rodriguez         

The first author of that disastrous publication, Julia Santiago, is currently tenure-track professor in Switzerland, her chair is financed by the Sandoz Family Foundation. The Santiago lab is also funded since 2017 by a €1.5 million ERC Starting Grant. I informed the ERC and Santiago’s new employer, University of Lausanne. ERC replied:

“The ERC will examine this information and address it within its applicable legal and procedural framework.“

The Naughty Pin Replicator

The University of Lausanne didn’t reply, and neither did Professor Santiago. At least her former postdoc advisor at the nearby University of Geneva, Michael Hothorn, shared his views on Twitter:

The 1st author of this work has completed two well-designed and carefully excuted studies as a postdoc in my lab, and has done very nice work of high technical standard as a PI. We do not know which author(s) were involved in the manipulations, so pls innocent until proven guilty https://t.co/b6Hthqck90

— Michael Hothorn (@structplantbio) June 24, 2021

Here are more papers of Dr Santiago with Prof Rodriguez. Like this one:

The last author José Márquez, who did a PhD in Valencia and is now team leader at EMBL in Grenoble, France, commented on PubPeer:

“The experiments in question were carried out in the laboratory of the corresponding author, Pedro Luis Rodriguez. I recently became aware of several other claims concerning the same group. I’ll be contacting him for information. “

Or this paper from the Rodriguez lab, Elisabeth Bik even included it in her popular Twitter “Spot the Problem” contest:

#ImageForensics
More baby plants, from the same group as my previous tweets.
Can you spot the problem here? Cited by 252 other papers. pic.twitter.com/nOkNDwypqK

— Elisabeth Bik (@MicrobiomDigest) June 24, 2021

Silvia Rubio, Americo Rodrigues, Angela Saez, Marie B. Dizon, Alexander Galle , Tae-Houn Kim , Julia Santiago , Jaume Flexas , Julian I. Schroeder , Pedro L. Rodriguez Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid    PLANT PHYSIOLOGY (2009) doi: 10.1104/pp.109.137174

This is what Bik’s followers found, a flipped segment with digitally cloned baby plants:

So much to analyse in that little dish:

Or, also very bad, this paper with many recurrent author names:

Rodriguez commented:

“We are currently checking our pin replicator. It will take two weeks to check whether pins can generate such problem It is quite difficult to localize authors responsible of figure preparation after 13 years Indeed, one author of this paper passed away last year“

But then Cheshire aka Actinopolyspora Biskrensis chimed in:

Elisabeth Bik found something as well, in Figure 4H: “Some of the baby plant parts appear to look more similar than expected.“

Rodriguez then pleaded:

“Please, we need some time to check the issue about the replicator. Finally, if these data are misrepresented, we will repeat the experiments and send the corresponding post Unfortunately, we do not have the original images after 13 y, and I do not have access to forensic tools as you are using here…”

There is of course more, even without Santiago. The following paper also fell prey to a malicious pin replicator.

Lesia Rodriguez , Miguel Gonzalez-Guzman, Maira Diaz, Americo Rodrigues, Ana C. Izquierdo-Garcia, Marta Peirats-Llobet, Maria A. Fernandez, Regina Antoni, Daniel Fernandez, Jose A. Marquez, Jose M. Mulet, Armando Albert, Pedro L. Rodriguez C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis The Plant Cell (2014) doi: 10.1105/tpc.114.129973 

Pedro Rodriguez, for some reason commenting as the first author Lesia Rodriguez Solovey (no relation), explained:

“I am not satisfied with the whole figure and we are going to repeat the experiments. Unfortunately, one key author of this paper passed away last year […reference to the late Portuguese professor Américo Rodrigues, -LS…], so we are missing key information. Yeast two hybrid assays of this paper were done several years before, probably by the same person who was involved in Santiago et al., 2009. I am not aware that journals request raw data for 10 y (even tax declaration only requires 5 y). We are not immune to informatic problems. One of the authors involved in the preparation of the figures will explain it. However, we maintain glycerol stocks (even more than 10 years!) and we can repeat the experiments.“

I’m not sure what the point of repeating the experiment is, when such “informatic” problems are found. Are Rodriguez’ students allowed to sit the exact same written exam again after they were caught cheating in it? I guess not? So why is this Dr Informatic allowed to do that?

In this collaborative paper, Rodriguez’ Chinese colleagues admitted a mistake in Figure 2A:

But whose mistake was the following (found by Cheshire), also Qian’s? Fig 4D shows not just duplicate images, they were also darkened, shifted or (yellow boxes) rotated. Honest mistake, really?

Indeed, Rodriguez hints on PubPeer that the Chinese colleagues are responsible:

“The figures/data in question did not originate from my laboratory“

But:

“I can understand that minor mistakes occur during the preparation of the figure, which do not affect the conclusions of the work.“

How can he be so sure then?

Honest mistakes

Can a pin replicator replicate pictures of plants and western blot bands? An academic question also for this collaborative study with Rodriguez colleague at National Center for Biotechnology in Madrid, Vicente Rubio:

María Luisa Irigoyen, Elisa Iniesto, Lesia Rodriguez, María Isabel Puga, Yuki Yanagawa, Elah Pick, Elizabeth Strickland, Javier Paz-Ares, Ning Wei, Geert De Jaeger, Pedro L. Rodriguez, Xing Wang Deng, Vicente Rubio Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis The Plant Cell (2014) doi: 10.1105/tpc.113.122234 

Rodriguez commented in this regard:

“The figures/data in question did not originate from my laboratory and I do not have copies of underlying raw data used for the figure preparation“

More image reuse, this time likely in Rodriguez’ own lab, also spotted by Bik:

Miguel Gonzalez-Guzman, Gaston A. Pizzio, Regina Antoni, Francisco Vera-Sirera, Ebe Merilo, George W. Bassel, Maria A. Fernández, Michael J. Holdsworth, Miguel Angel Perez-Amador, Hannes Kollist, Pedro L. Rodriguez Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid The Plant Cell (2012) doi: 10.1105/tpc.112.098574 

Regarding the pentuplicated RT-PCR gel experiment in Fig 3A, Rodriguez stated on PubPeer:

“I really do not know if the background should be different, particularly in the darker version you show.”

Can someone show the professor how DNA gels work?

Anyway, this all must have been an “honest mistake”, given how Rodriguez describes for the next paper the missteps of his PhD student Miguel Gonzalez-Guzman, who is now investigator at the University Jaume I, also in Valencia:

Miguel González-Guzmán, David Abia , Julio Salinas , Ramón Serrano, Pedro L. Rodríguez Two new alleles of the abscisic aldehyde oxidase 3 gene reveal its role in abscisic acid biosynthesis in seeds PLANT PHYSIOLOGY (2004) doi: 10.1104/pp.103.036590 

Rodriguez shared with me a reply to “Dear Aneurus Inconstans” which Gonzalez-Guzman planned to post on PubPeer (here it is now). It is indeed tricky to explain. The “MS” images for Col on the left-hand side are identical also, but the NACl and Mannitol samples got swapped between papers. It seems the authors split one experiment into two publications, which is OK in itself, but not OK is that they apparently only ever took one close-up picture for each plant genotype, experimental condition and time point, and called it representative. As Rodriguez informed me, “aba2-11 is another name for sre1“, so the image re-use for another salt-resistant mutant, sre2-2, was not appropriate.

The penultimate author Ramon Serrano is Rodriguez’ former mentor and now emeritus professor at the same IBMCP in Valencia. He and Gonzalez-Guzman are also on this paper:

Silvia Rubio, Tony R. Larson, Miguel Gonzalez-Guzman, Santiago Alejandro, Ian A. Graham, Ramón Serrano, Pedro L. Rodriguez An Arabidopsis mutant impaired in coenzyme A biosynthesis is sugar dependent for seedling establishment PLANT PHYSIOLOGY (2006) doi: 10.1104/pp.105.072066 

Further analysis by A, Inconstans showed that the gel bands seem indeed duplicated. Rodriguez commented on PubPeer:

“After receiving your comments, I have realized that a checking point of figures with forensic tools is required. I would say this should be performed by journals before final acceptance, indeed that is the case for some of them.“

I’m not sure the journal editors will agree that the blame lies on them. Sure, journals should be checking papers for data manipulations, but AFAIK it is authors’ own responsibility not to fake data in the first place.

Rodriguez replied the same way on another PubPeer thread. This was the oldest paper flagged so far:

Rodriguez commented on PubPeer:

“I can understand that DNA bands look very similar in the black/white negative, but I do not have forensic tools as you have. Anyway, this figure is redundant with panel B. You are raising an important question in scientific publications.“

Followed again by the disappointment that the journal editors didn’t catch that.

Kudla in Münster

Aneurus Inconstans stumbled across Rodriguez’ papers while analysing the works of someone else, namely of Jörg Kudla, professor at University of Münster in Germany, who now has 9 papers flagged on PubPeer. Here is that collaborative paper between Rodriguez and Kudla:

Borja Belda‐Palazon, Jose Julian, Alberto Coego, Qian Wu, Xu Zhang, Oliver Batistic, Saleh A. Alquraishi, Joerg Kudla, Chengcai An, Pedro L. Rodriguez ABA inhibits myristoylation and induces shuttling of the RGLG 1 E3 ligase to promote nuclear degradation of PP 2 CA The Plant journal (2019) doi: 10.1111/tpj.14274

An anonymous commenter, who later turned out to be Rodriguez, wrote:

“The last yeast dilution (10-3) contains a low number of cells, which are applied using Boekel Scientific™ 48/96 Pin Replicators (not by hand), so there is a chance that some pins produce a similar pattern“

A. inconstans retorted:

Rodriguez was only partially swayed:

“Ok, thanks, then it might be a mistake (1 out of 72 panels) in the preparation of the figure Alternatively, some pins might produce a similar imprint because they are burned with ethanol to eliminate the yeast colonies.“

That naughty pin replicator again. And you know what? It even travelled to Münster to attack Kudla’s own research!

Üner Kolukisaoglu, Stefan Weinl, Dragica Blazevic, Oliver Batistic, Jörg Kudla Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks PLANT PHYSIOLOGY (2004) doi: 10.1104/pp.103.033068 

Also Kudla admitted the duplications, but:

“The marked panels of yeast colonies are identical. This mistake does not affect the conclusion of this experiment […] Moreover, several of the reported CBL1 and/or CBL9-CIPK interactions have been independently confirmed in a number of subsequent publications“

I informed the publisher and the University of Münster. The former promised an investigation, the Ombudsman of the latter informed me:

“Prof. Kudla has informed me that the scientific integrity and conclusions of the publications you mentioned are in no way objectionable, but that there have been errors – especially in the representations – which require clarification and correction. The correction process was initiated long before your complaints were received [this paper and most others were first flagged by A. inconstans on PubPeer 21-26 May 2021. I informed the university on 27 May, -LS]. The correspondence with the magazines in which the corrections are to be printed has been forwarded to me. In essence, the correction process has already been completed. Some corrections have also already been made to PubPeer. Only one publication is still open in the journal Plant Physiology.[…]

In view of this situation, I see no reason to forward the matter to the investigative commission of our university for the procedure in the event of suspected scientific misconduct.“

Kudla never replied to my emails. I understand, I am not popular with Münster professors, after the Püschel and Schwamborn affair.

Münster charges Jens Schwamborn’s mentor Andreas Püschel with research misconduct, again

“I am not under investigation“

But I exchanged many emails with Rodriguez. Here some excerpts:

“PIs usually trust in the work of students, who are excellent persons but sometimes make mistakes (as we do because even scientists are human beings). Most of the problems detected by Pubpeer (in my case) are the loading controls of drop tests. I have original files for some of these experiments and I do not understand why the students have chosen wrong yeast strips. I could speculate, but this is not helpful

Anyway, I am aware I face a difficult time and I will contact editors to explain the problems detected by Pubpeer. In the meantime we are repeating some of these assays and we are checking the original files when possible […]

Perhaps I trusted too much in figure preparation (this is a duty for PhD training), which I have improved during my last years.

Usually the pubpeer analyses are focused on relatively old articles, so it is difficult the correction of mistakes Two minor questions raised on recent articles have been corrected and in one case the correction was posted (https://doi.org/10.1105/tpc.20.00781). So, we need time to check the other problems […]

I am not under investigation. I will contact the editors of the journals and I will explain the problems detected.
Then, we will see what happens. I can take full responsibility if results obtained in my lab are not reproducible or if our genetic resources are not available for other people, for example. But fortunately, this never happened in my career, indeed we have provided a rich source of materials that were very useful to other scientists“.

The Plant Journal informed that they are processing Rodriguez’ retraction request. A good start. In his last email to me, Rodriguez wrote:

“If you do not accept honest mistakes, you destroy science. Your blind fury against wrong images does not consider the whole message and prevents a reasonable explanation
Come on, you want a better science, but this looks like the Spanish Inquisition“

Nobody expects the Spanish Inquisition.

This article was slightly updated soon after it first appeared, e.g. due to new PubPeer comments by Rodriguez.

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