Steffen Reinbothe: duplications planted on PNAS contributed track

This is a story of a plant scientist in France. No, not Olivier Voinnet or any of his Strasbourg associates. This time it is about Steffen Reinbothe, a German researcher specialising on chlorophyll synthesis and chloroplast development, whose discoveries made him appreciated in a very particular way among his peers, and whose research findings were so amazing that only the most skilled of researchers (i.e., himself) could reproduce them. Steffen and his sister Christiane Reinbothe used to hold academic positions in Germany, but they preferred to take residence in Grenoble, France. A big loss for German science, which might have had to do with a dossier from 2009, made by a former lab member and circulated among peers. It discussed the somewhat unorthodox way of figure assembly and gel data presentation employed by Dr Reinbothe and his coauthors. The old dossier is complemented with some oldish PubPeer evidence and newly made finds.

Whatever concerns any peers might have had about the reliability of their data: Steffen and Christiane Reinbothe could rely on the notorious “contributed” track at PNAS. It sure saw their academic careers through a long dry spell, and the journal played game regardless of anything.

Steffen Reinbothe used to be a bigwig in plant sciences, but it got quiet around him and his sister now. The journal venues are less impressive, even PNAS “contributed” track has likely ended, strangely not because of the excessive and embarrassing overuse, but due to the death of their “contributing” patron. There were better days: a paper in Nature which peers could make no head or tail of, and publications in other elite journals, like The Plant Journal, Reinbothe et al 2005, as discussed on PubPeer.

Let us ignore for now the two gels on the right, where the last empty lane was stuck on (we must of course trust Reinbothes that it is perfectly correct and not fishy at all). But what about the two gels on the left? They are surprisingly similar, down to the most minute detail, i.e. except of one single band in the 3rd lane, which does not at all look like it was sloppily stuck on in Photoshop, with its edge outside the actual gel lane. The journal’s editors were aware of that since March 2015, and seem unconcerned. Maybe we should trust the academic authorities also here.

The reader from Sweden

The Reinbothe dossier contains additional material to what is already publicly on PubPeer, and is available here. It was forwarded to me by a reader of my site from Sweden. The reader tells this story:

“It dates back to 1999 when I did my diploma work in plant physiology. My supervisor, then a PhD student now a full professor and my department head, was busy trying to replicate some experiments of the Reinbothe group. The group in Gothenburg had a long history of working with pigment protein complexes and formation of chlorophyll in plants following exposure to light. In particular they were interested in a protein called protochlorophyllide oxidoreductase (POR) that converts protochlorophyllide into chlorophyllide; a step in the synthesis of chlorophyll. Interestingly this enzyme is activated by light absorption by its very substrate. This is fairly unusual among enzymes. In the absence of light the internal structures of the chloroplasts does not quite form, instead a complex membrane bound structure containing complexes of POR and protochlorophyllide forms. A short flash of light triggers the immediate conversion of protochlorophyllide to chlorophyllide and the subsequent dissolution of the so called prolamellar body.

The group in Gothenburg under, the now late, professor Christer Sundqvist was specialized in the different POR complexes and their spectral characteristics. POR is protein encoded by the nuclear genome, and it is thus imported into the chloroplast after translation on cytosolic ribosomes. Reinbothe claimed that at least one of the POR forms followed a special import route and that the import was stimulated by the presence of the substrate protochlorophyllide in the chloroplast stroma. A somewhat exiting finding. In particular the presence of a second protein import apparatus in the chloroplast envelope would have been big news in the late nineties. Only problem is that it was impossible to reproduce. On associate professor from Gothenburg as I heard went to visit Reinbothe to witness the magic first hand. He came back with the following summary:

“The results were only reproducible if Steffen was alone with the blots in the darkroom”.

Not much actually happened after that except that the PhD student Henrik Aronsson published a couple of different papers essentially showing non-reproducibility of Reinbothes work on import of POR into chloroplasts (Aronsson et al 2000, Dahlin et al 2001, Aronsson et al 2003, Aronsson et al 2008). There was a paper from the Reinbothe group published in Nature (Reinbothe, Lebedev & Reinbothe 1999) which I never could quite understand but Christer genuinely thought it was total bogus and there was something of a rebuttal published a little later (Armstrong et al, Trends in Plant Science, 2000). Christer is not listed as an author though, but I believe he did contribute quite a lot to that manuscript. 

I personally went on to a PhD in another plant science field in the same department and then I did not hear much until 2010 when Henrik received the anonymous file from someone in Reinbothes lab. I know that Henrik had some kind of contact with some German professor about a formal investigation into the matter, but as far as I know nothing apparently happened. All I have seen is a pretty steady stream of papers in chiefly PNAS but also other good plant journals”.

GDR to Grenoble

There are no proper CVs of Reinbothe online, so one can only try to reconstruct. He was born in 1962, meaning he is 56 now and can soon quietly retire as emeritus professor according to French pension rules. PubPeer gives a clue where the academic career of Steffen Reinbothe has started, in the lab of Benno Parthier at the University of Halle, Germany, back then in the socialist Eastern Germany (GDR). As the two Germanies united to form one, a gel split itself in two, to appear in two separate papers, showing two separate experiments in Reinbothe et al 1990 and Reinbothe et al 1993, as evidenced on PubPeer:

Then somehow, the Reinbothe siblings went to Grenoble in France, and stayed there until this very day, with a short pop over back to Germany. The Reinbothes are truly a perfect fit for French plant sciences, as readers of my site might appreciate.

When the 1999 Nature paper was published, both Reinbothes, Steffen and his sister Christiane, had as affiliation the French Centre National de la Recherche Scientifique (CNRS) and its Centre d’Etudes et de Recherches sur les Molécules Organisées (CERMO), and Universite Joseph Fourier (now Universite Grenoble Alpes) in Grenoble. A January 1999 press release from the University of Bochum in Germany, celebrating that Nature paper, describes Steffen Reinbothe as a “New-Bochumer”, funded with the Heisenberg fellowship by the DFG, which would mean he was about to be appointed as full professor there. DFG shows no records for Heisenberg funding to Reinbothe though, only a “material grant”. The same kind of professorial fellowship the diabetes researcher Kathrin Maedler in Bremen agreed to resign from last year, after DFG investigation.

In 2000, obviously because of the Nature paper, Steffen Reinbothe was appointed associate professor in Grenoble, and full professor in 2002. Christiane Reinbothe used to be affiliated with the University of Bayreuth, also in Germany. She left Bayreuth in 2005 to follow a “Chaire d’Excellence at the University of Grenoble I”. In 2006, she was appointed professor in Grenoble for a period of 3 years. We do not know how the evaluation went then, the Universite Grenoble Alpes has no public online records of Christiane now, and only one information-free entry for Steffen.  Since 2018, Christiane Reinbothe reverted to using her long-outdated Bayreuth institutional email address, but with Grenoble affiliation, as indicated in this recent preprint on BioRxiv.

We do not know how big the Steffen and Christiane Reinbothe lab is, and how many young researchers are being trained there in the digital art of creative plant sciences. French records show that Steffen Reinbothe up to December 2017 supervised exactly 2 PhD dissertations. One of those two was by Edouard Boex-Fontvieille, supervised jointly together with Christiane Reinbothe. Despite such smallish lab, the sibling team is still incredibly productive, churning out several papers a year even now, including in the self-proclaimed “leading journal in its field”, Frontiers in Plant Science. Even that paper, Boex-Fontvieille et al 2006 contains what looks like a duplication of loading controls in Figure 5:

I was joking of course, Frontiers in Plant Science, like all Frontiers journals, is not really “a leading journal in its field”, unless the field leadership is defined by the open access publication charges. Reinbothes, or rather their University of Grenoble, had to cough up $2950 to publish the above shown duplciated figure there. The next year after the Frontiers success, Reinbothes placed this in Plant Molecular Biology, Buhr et al 2017:

Maybe the gel apparatus in Reinbothes’ Grenoble lab malfunctioned, once again. Somehow the whole block with five gel lanes got duplicated, twice, in both Figure 2 panels B and C. This is what apparently happens if you load 5 “transformants” on your gel and leave the lab a for a coffee break: you come back to find 10 of them, as a 1:1 copy.

Contributing patron

Those minor problems aside, what real treasure Reinbothes do have, or maybe used to have, was a special kind of subscription to the elite US journal PNAS (Proceedings of National Academy of Sciences, USA). Between 1994 and 2017, the siblings published impressive 19 papers there, all of which were “contributed” or “communicated” by the US-based plant biologist Diter von Wettstein, who died in 2017, aged 88. That peculiar track is unique to PNAS and available only to members of the US National Academy of Sciences, as the late von Wettstein used to be.

The PNAS loophole basically means the “contributed” or “communicated” papers bypass editorial oversight and proper peer review, since the NAS member authors arrange all of it themselves. The “communicated” track, where NAS members were sneaking in papers they were themselves not authors on, was shut down in 2010. Apparently, the NAS members had enough being pestered for favours without getting anything in return. But the “contributed” track remains, NAS members sure wanted to keep that privilege to publish their own research (or their unpublishable bunk) outside peer review. Since then, the ancient von Wettstein was always the “contributing” co-author on Reinbothes’ papers. 19 PNAS papers in 13 years, not many plant scientists can pull this off. Who will “contribute” Reinbothes’ papers to PNAS now?

The dossier, assembled in 2009, deals with some of those PNAS papers.

Minor duplications

On several occasions, datasets from different PNAS papers, presented as being from separate experimental studies, seem duplicated. The dossier visualises that, for Reinbothe et al 2004 and Reinbothe et al 2006:

Basically, the two figures differ in principle only on one new dataset, IEP36, and the strange case of pPORA samples running differently between the two figures. Maybe the pPORA Excel data rotted somewhat during those 2 years of storage on Reinbothe’s computer.

The most problematic paper however is this one:

Stephan Pollmann, Armin Springer, Frank Buhr, Abder Lahroussi, Iga Samol, Jean-Marc Bonneville, Gabrielle Tichtinsky, Diter von Wettstein, Christiane Reinbothe, and Steffen Reinbothe

A plant porphyria related to defects in plastid import of protochlorophyllide oxidoreductase A

Proc Natl Acad Sci USA 2007 DOI:  10.1073/pnas.0610934104

The first author Stephan Pollmann did his PhD in Bochum and thanked Steffen Reinbothe in his 2002 thesis for a “truly “masterly” instructing on two-dimensional gel electrophoresis“. The irony is strong here. Now Pollman teaches those amazing gel skills to his lab members in Spain (another great loss for German plant sciences), luckily there are presently just two of them: a postdoc and a technician.

The Pollmann et al 2007 paper apparently recycles its own data and even makes use of results from another Reinbothes paper, Reinbothe et al PNAS 2000, published 7 years earlier.

As above, some experimentally utterly distinct and unrelated datasets mysteriously produce exactly same graphs, which neatly overlap.

That Pollmann et al PNAS 2007 paper holds more surprises. Figure 6B purportedly showing a blue light gel image and a western blot of same gel suggests that the authors not only transferred the protein band very faithfully, but also the minute image acquisition artefacts from gel picture to blot (similar thing happened in Buhr et al PNAS 2008, see dossier).

Another set of bands shown in Pollmann et al 2007 used to exist before, in a different Reinbothe paper: Reinbothe et al, Mol Genet Genomics, 2006. The gel picture fell on its head while travelling and became very negative about it, it seems.

Applied democracy

The Pollmann et al PNAS 2007 paper received two interesting corrections in 2010, apparently after the 2009 dossier I now make public was circulated in the plant science community. The first correction was signed by the paper’s coauthors and Reinbothes’ University of Grenoble colleagues Jean-Marc Bonneville and Gabrielle Tichtinsky, and went like this:

“The undersigned authors wish to note that: “We have been associated with part of the experimental work described therein, yet now disagree with the title and main conclusion of the article. […] We were never granted access to the complete set of original data, in particular to those supporting Figs. 2G, 4 C and D, and 6B. Therefore, we wonder about the quality of the underlying work and have expressed doubts concerning what is concluded from these pictures. It must also be made clear that the flu mutant used is not a published allele but contains a T-DNA insertion (SALK_002383) and displays a weak phenotype”

The second correction was signed by all the other coauthors, led by Pollman, the Reinbothes and their patron von Wettstein:

“The undersigned authors wish to note that: “We confirm that we are confident in the data and conclusions in the published article.”

This was how the elite journal PNAS democratically resolved the issue of data integrity: the majority of 8 undersigned authors won against their 2 pathetic critics, with an 80% landslide majority. The paper now proven as reliable, science once again self-corrected itself.

Even after that incident the Reinbothes were invited to submit 6 more papers to PNAS, again all “contributed” by von Wettstein. One of them, Springer et al 2016, contains such an interesting Figure 3, as evidenced on PubPeer:

Each panel is a composite, the bands were sliced and spliced. Presented as a continuous two-lane gel, so the duped reader can appreciate the absence of signal in one lane and the band in the next one. Quality science contributed to PNAS on behalf of Reinbothes by the 87-year old von Wettstein, one year before his death.

If any obvious lessons are to be drawn from the Reinbothe case, it is that science is the most tolerant of societies: there is indeed a place with second chances for everyone, provided they maintain both friendships and publishing at a decent level. This we already learned in another plant science case, with Olivier Voinnet. And thanks to PNAS contributed track, Reinbothes published rather decently, until last year.

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