Journal of Experimental Botanydoi:10.1093/jxb/ert461

Flowering newsletter review

My favourite flowering image: a cob of pod corn

Günter Theißen*

Department of Genetics, Friedrich Schiller University Jena, Philosophenweg 12, D-07743 Jena, Germany

* To whom correspondence should be addressed. E-mail: guenter.theissen@uni-jena.de

Received 6 December 2013; Revised 18 November 2013; Accepted 29 November 2013

Abstract

For good reasons scientists usually do not report the personal circumstances of their work when publishing their results. This means, however, that the scientific facts being reported may not accurately reflect the personal impor-tance of the respective work for the individual scientists. Pictures of pod corn (or Tunicate maize) have been on my mind for much of my life, through good and through bad times. This is why…

Key words: Domestication, inflated calyx syndrome, MADS-box gene, maize, STMADS11-like gene, SVP, Tunicate mutant, Zea mays.

I would probably never have been obsessed by pod corn without my father. When he became chronically ill in the early 1970s he started reading frantically a wide variety of books. One of these books (Grebenscikov, 1959) was about the domestication of maize (Zea mays ssp. mays). In contrast to the strange novels my father was reading, it immediately caught my interest. I was impressed by the enormous agronomic and religious impor-tance of maize for many Native American Indian tribes. I was even more provoked by the fact that maize does not exist in the wild. [We now know that maize is morphologically so differ-ent from its wild ancestor, teosinte (Zea mays ssp. parviglumis) that ancestry is not easily recognizable.] I was most fascinated, however, by a picture of a strange mutant of maize, pod corn (Tunicate maize) that was, at this time, hypothesized to repre-sent an ancestral form of maize (Fig. 22 in Grebenscikov, 1959). Due to its amazing phenotype, pod corn has been of religious importance for certain Native American Indian tribes since pre-Columbian times, who believed in its curative and magical powers (Cutler, 1944; Mangelsdorf, 1948, 1958). It seemed that I was not the only one who was fascinated by pod corn.

The most prominent phenotypic feature of pod corn is a foliaceous elongation of the glumes, such that they cover the kernels in the ears (Fig. 1; cob on the right). This is different from ordinary maize varieties in which glumes are not present or are so short that they are invisible in the mature ear (Fig. 1; cob on the left).

As a postdoctoral student in the early 1990s, I seriously considered continuing my work as a molecular biologist to try to work for the benefit of mankind by helping to cure AIDS or other devastating diseases (‘It’s the economy, stupid!’). However, when Heinz Saedler at the Max Planck Institute for Breeding Research in Cologne offered me the chance to work on the domestication of maize, I remem-bered that strange book of my father’s and couldn’t resist the opportunity. Among a collection of maize cobs in Heinz’s office I  saw the ‘real thing’ for the first time. I  was impressed again. It didn’t look like one of those usual mutants where one immediately gets the feeling that there is something completely wrong. On the contrary, the mutant phenotype was of bizarre beauty and appeared quite functional. There is nothing wrong with wrapping fruits in long glumes, I thought, almost all grasses do this. Suddenly wild-type maize with its naked kernels appeared stranger to me than the Tunicate mutant. (One should say that only the cobs of heterozygous plants are appeal-ing; mutants homozygous for the Tunicate allele are less impressive.)

After some initial detours, I  started a project in Heinz’s department studying the contribution of MADS-box genes to the domestication of maize and their potential use for rational crop plant design. cDNAs and genomic fragments of these genes were cloned and their chromosomal map positions were

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determined in order to see whether they mapped to any of the chromosomal regions that John Doebley and his coworkers had identified as the regions relevant for the origin of maize (Doebley et al., 1990). This naïve candidate gene approach was initially lots of fun but, in the long run, exhausting, not least because we had initially underestimated the total number of MADS-box genes in maize by about an order of magnitude.

One day in the late 1990s we got a new set of mapping data. Thomas Münster, a postdoctoral student in the laboratory, told me that, yet again, none of our genes mapped to one of the ‘Doebley regions’. He also told me that one of the genes mapped closely to a mutant of maize termed ‘Tunicate’ and he asked me whether this made any sense to me. It did! I knew that Tunicate is based on a dominant, gain-of-function muta-tion and that many of these mutations bring about their effects because of ectopic expression of the gene, so I  pre-dicted that the Tunicate gene is expressed in the glumes of maize in the Tunicate mutant, but not in the wild type.

It took me a while to convince someone in the laboratory to work on Tunicate, since such a project did not follow our

major goals. Luzie Wingen, a PhD student in my group at that time, eventually agreed to embark on this endeavor. She just needed a few weeks to grow the plants and to do some initial Northern hybridization experiments. The results were clear-cut: in all of the wild-type plants she tested, the gene was expressed only in vegetative tissues, especially in leaves, while in the Tunicate mutant there was strong ectopic expression in male and female inflorescences. Luzie confirmed her findings by detailed in situ hybridization experiments. Since ectopic expression was not observed for other, closely related genes, we were convinced that we had cloned the Tunicate gene.

The mapping result was also interesting for another reason: it associated a mutant phenotype and hence a function with a new subfamily of MADS-box genes that was unknown at that time. Wolfram Faigl, a technician in our laboratory, and Thomas had cloned several subfamily members from maize and termed them ‘Faimadse’. When the first subfamily mem-ber was published from potato as STMADS11 (Carmona et al., 1998), we applied the priority rule for naming clades of MADS-box genes (Theißen et al., 1996; Becker and Theißen, 2003) and termed the whole clade ‘STMADS11-like genes’ (Becker et  al., 2000; Becker and Theißen, 2003). The gene SHORT VEGETATIVE PHASE (SVP) from Arabidopsis thaliana, cloned by Peter Huijser and co-workers (Hartmann et  al., 2000), is probably the best-known STMADS11-like gene. It turned out that STMADS11-like genes have diverse and interesting functions in phase transitions and other devel-opmental processes and hence they have become the subject of intense research to the present day (Khan and Ali, 2013).

Following a tradition in our laboratory (Theißen et  al., 1995), the Tunicate candidate gene was termed ZMM19. Further work corroborated our hypothesis that ZMM19 represents the Tunicate locus. We found, for example, that ZMM19 is a duplicate locus in Tunicate plants but not in wild-type plants and has rearrangements in the promoter regions of the mutant. These findings fitted nicely to some classical articles on maize genetics maintaining that the mutant Tu locus is complex and compound (Mangelsdorf and Galinat, 1964).

In March 2000, Luzie presented our results at the 42nd Annual Maize Genetics Conference in Cœur d’Alene, Idaho, USA (Wingen et  al., 2000). In a detailed report about this conference, Running et  al. (2000) devoted a whole sec-tion to our findings, starting with the statement that ‘Luzie Wingen…described the cloning of the Tunicate1 (tu1) gene’. I was convinced that we would publish our findings within a few months but I was wrong.

We decided to do some additional experiments to corrobo-rate our findings. For example, we cloned and characterized some ‘weak’ Tunicate alleles with the help of Hans Sommer. Our additional experiments all finally worked out nicely, but slowly. The fact that some of us, including Luzie and me, left Cologne and moved on to new challenging and time-consum-ing jobs did not speed up the project either.

Only a few brief messages were sent to the world about our findings. In a paper in Maydica about MADS-box genes in maize, we reported that ZMM19 mapped closely to the Tunicate locus and that several other independent lines of

Fig. 1. An ear from ‘wild type’ maize (left) and pod corn (right). The pod corn ear is from a heterozygous Tu/+ plant; homozygous plants have an even stronger mutant phenotype, but have, in the author’s view, less beauty. (Picture taken from Wingen et al., 2012. The molecular basis of pod corn (Tunicate maize). Proceedings of the National Academy of Sciences, USA 109, 7115–7120.)

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evidence strongly supported the view that ZMM19 indeed represented the Tunicate gene (Münster et  al., 2002). A  bit later, Chaoying He, Thomas Münster, and Heinz summarized our findings in an article about ‘evolutionary novelties’ (He et al., 2004). The piece was termed a ‘minireview’ but, in the case of the Tunicate story, it reported results almost a decade before they were actually published so that ‘preview’ would have been a more appropriate term. In the same year we sub-mitted the sequence of ZMM19 from Tunicate to GenBank. This obviously helped others to uncover the molecular basis of the Tunicate gene (see Acknowledgements in Han et al., 2012).

Even though most of our data on Tunicate were not yet published, they nevertheless inspired others to look for simi-lar phenomena elsewhere. Heinz wondered whether ortho-logues of a gene that makes glumes elongate upon ectopic expression may have a similar effect on sepals. Along these lines of thought he started to investigate the ‘inflated calyx’ trait of the Solanaceae. Heinz’s intuition was right and, since he had teamed up with co-workers that were on a faster pub-lication track, the initial work on the inflated calyx phenom-enon was published many years before our work on Tunicate (He and Saedler, 2005).

While the inflated calyx story generated a tsunami of publications (He and Saedler, 2007; He et al., 2007; Hu and Saedler, 2007; Khan et al., 2009, 2012; Zhang et al., 2012), the original source of inspiration made little waves. That we may have missed a chance to get a good publication was therefore hard to deny. Nevertheless, it took us a while to draft the first manuscript as most of us were busy with other duties that appeared more important at that time. Since I  still thought that we had cloned one of the coolest genes on this planet we decided, in 2005, to send the manuscript to one of these prestigious journals you all know (journal title available upon request). However, reviewers told us that our manuscript was ‘just a cloning paper’ and thus only of limited interest. I felt I was still playing Classic Rock (gene cloning) at a time when everyone was already listening to Hip Hop and House (genomics). I actually wanted proudly to present the paper to my father, but time was running out: After decades of illness he died at the end of 2005 before our work was published.

For the reasons mentioned before it took us quite some time to rewrite the manuscript and to send it to the next pres-tigious journal. This time—in 2008—the Editor did not even send it out for review (Classic Rock was only a faint memory by then).

But I  couldn’t get these pictures of pod corn out of my mind. Luzie also started to push me. When I learned that the 52nd Annual Maize Genetics Conference was to be held in Europe for the first time and at such a nice location as Riva at the northern end of Lake Garda in Italy, I decided to adver-tise our work on Tunicate once again. This time, 10 years after Luzie’s presentation in Idaho, a poster presentation was all I could get, but I was more than pleased. In Riva, I met Jong-Jin Han from Rob Martienssen’s group at the Cold Spring Harbor Laboratory in New York. I learned that he was also working on Tunicate and that he had for quite a while hypoth-esized that Tunicate encodes a microRNA but he was now

also convinced that the MADS-box gene ZMM19 represents the Tunicate locus.

Being confirmed in this way is somehow a mixed blessing. We gave the prestigious journal approach a final try and sent the manuscript to PNAS in 2011. This time we were lucky in that John Doebley got to handle it—if there is anyone on this planet that has sympathy for work on maize, it is prob-ably him, and, amazingly, one of our reviewers remembered Luzie’s talk 12 years ago and strongly argued that publication of our data was overdue. That was hard to deny. All in all, the reviewer’s requests were tough, but fair, and thus revision of the manuscript was hard and time-consuming, but do-able. The letter explaining our changes to the manuscript that Luzie and I produced was finally almost as long as the whole manuscript.

During that time my mother suddenly became very ill. Thus I had to do much of the work required for revision and the correction of the galley proofs during long train rides. At that time I often felt that working on bizarre plant mutants doesn’t make any sense at all, because it doesn’t help to reduce misery and pain. One day I was sitting at my mother’s bed that she could not leave anymore with a version of the paper published online ahead of print. I wanted to show it to her but she was too weak to recognize what it was about. She probably would not have been interested in it anymore anyway.

About 15 years had passed since we had cloned the Tunicate gene, and I should have been happy that we finally managed to get it published in a decent journal. Mission accomplished! But I felt nothing but emptiness. Our paper was finally pub-lished in print on the first of May 2012 (Wingen et al., 2012). All’s well that ends well! Two days later my mother died.PS: Jong-Jin’s very detailed work was published two

months later in The Plant Cell (Han et al., 2012)—he even got the cover! The paper not only confirmed all of our key findings but also had a number of interesting new results to report. For example, the authors demonstrated that the rear-rangement in the promoter region of ZMM19 in Tunicate is based on a 1.8 Mb chromosomal inversion that fused a gene expressed in inflorescences to the 5’ regulatory region of ZMM19; that the two ZMM19 copies in Tunicate are about 30 kb apart (we had wondered about this for a long time); and that transgenic lines in which ZMM19 expression is driven by the rearranged promoter region phenocopy the Tunicate trait.Addendum: I am fully aware that the picture I have chosen

does not show a flower, but two infructescences. I apologize for any confusion that this may cause. My only excuse: There is just no flower picture that means so much to me.

Acknowledgements

I thank Wim Deleu and Hans Sommer for their contribu-tions to the Tunicate project. Special thanks go to Wolfram Faigl and Thomas Münster for playing Klotto and pick-ing Faimadse clone wf4123, that was renamed into SUPER TECHNICAL ASSISTANT GENE1 (STAG1), that was renamed into TM078, that was renamed into ZMM19,

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and eventually turned out to be the TUNICATE gene. I am especially grateful to Heinz Saedler without whose continu-ous support the project would not even have been initiated and to Luzie Wingen, for without her final enthusiasm for the Tunicate work our data may never have been published. I thank Jong-Jin Han for a long talk in Riva. I am indebted to John Doebley for giving us a chance. Many thanks also to Wolfram, Thomas, and Luzie for helpful comments on a pre-vious version of the manuscript. Special thanks go to Lydia Gramzow for improving my English and to Lars Hennig for inviting me to write about my favourite flower image and for his patience. This paper is dedicated to my parents, Heiner Theißen (1940–2005) and Marlies Theißen (1939–2012).

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