TOPLESS across plant evolution

Although plants and animals became multicellular organisms independently they use many of the same mechanisms to control the complex feat of going from one cell to trillion. Both need to produce organs (arms or leaves) and their embryos need to decide what is top and bottom (head vs feet or shoot vs root). After a round of random mutagenesis (Long et al., 2002; Long et al., 2006), identified a mutant, which at elevated temperatures had the human equivalent of two heads, one of them being where its legs/feet should be. This doubled rooted phenotype was caused by a mutation in a transcriptional co-repressors then named TOPLESS due to the lack of a ‘top’ (shoot) in the mutant plant. The proteins encoded by the TOPLESS gene in plants has related co-repressors in animals. Their job is to regulated which genes are turned off. But they do no bind DNA directly and have no innate specificity for particular genes. Specificity is achieved through transcription factors binding target genes and recruiting co-repressors, which mediates the repression of these genes. It is still not fully understood why the mutation in TOPLESS causes the double rooted phenotype as the TOPLESS protein is involved in many processes including regulating stem cell number and flowering time (Causier et al., 2012a). However, it probably involves the plant hormone auxin. This hormone has a role in nearly every aspect of plant development, including top-bottom (apical-basal) patterning. It has been shown TOPLESS is used by regulators of auxin-dependent gene expression (Aux/IAA proteins) to turn of genes (Szemenyei et al., 2008).

We decided to ask when TOPLESS become involved with auxin during the evolution of plants. The above work was done in the model flowering plant Arabidopsis thaliana. The first flowering plants emerged during the time of the dinosaurs (about 150 million years ago) but plants first appeared on land about half a billion years ago! One of these early groups of land plants resembled modern day mosses. Taking living mosses like the lab model Physcomitrella patens, we can ask if the same mechanisms controlling growth existed in the last common ancestor of mosses and flowering plants, which existed about half a billion years ago.

Using a technique called yeast-two hybrid we can detect if two proteins physically interact. We took the two moss TOPLESS proteins and three Aux/IAA proteins and tested their interaction. As expected, both TOPLESS proteins interacted with all three Aux/IAA proteins (Causier et al., 2012b). Previous work has shown A. thaliana TOPLESS interacts with Aux/IAA proteins by using their LxLxL motif in domain 1 (where L is Leucine and x is any amino acid). Moss TOPLESS proteins have an LxLxPP (P is Proline) instead and might be the functional equivalent (Paponov et al., 2009). We mutated the second L of the LxLxPP motif and found no interaction between TOPLESS proteins and Aux/IAA proteins (Causier et al., 2012b).

Previously, it was shown some auxin response factors (ARF proteins) from A. thaliana interact with TOPLESS (Causier et al., 2012a). These are not the ARFs involved with the ‘classical’ auxin signalling. These are repressors and little is know about them. This work suggests they may act as repressors thanks to the actions of TOPLESS. We investigated if these interactions were also conserved in moss. Although not all repressive ARFs in moss interacted with TOPLESS, two did (Causier et al., 2012b). These are form the ARF10/16/17 family.

Together, these data show that TOPLESS has been involved with auxin signalling components for the last 500 million years since the origins of land plants. Perhaps these interactions pre-date land plants? But we have shown using simple lab techniques we can try to answer fundamental questions about how the regulation of gene expression has evolved.

Causier B, Ashworth M, Guo W, & Davies B (2012). The TOPLESS interactome: a framework for gene repression in Arabidopsis. Plant physiology, 158 (1), 423-38 PMID: 22065421

Causier B, Lloyd J, Stevens L, & Davies B (2012). TOPLESS co-repressor interactions and their evolutionary conservation in plants. Plant signaling & behavior, 7 (3) PMID: 22476455

Long JA, Ohno C, Smith ZR, & Meyerowitz EM (2006). TOPLESS regulates apical embryonic fate in Arabidopsis. Science (New York, N.Y.), 312 (5779), 1520-3 PMID: 16763149

Long JA, Woody S, Poethig S, Meyerowitz EM, & Barton MK (2002). Transformation of shoots into roots in Arabidopsis embryos mutant at the TOPLESS locus. Development (Cambridge, England), 129 (12), 2797-806 PMID: 12050130

Paponov IA, Teale W, Lang D, Paponov M, Reski R, Rensing SA, & Palme K (2009). The evolution of nuclear auxin signalling. BMC evolutionary biology, 9 PMID: 19493348

Szemenyei H, Hannon M, & Long JA (2008). TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science (New York, N.Y.), 319 (5868), 1384-6 PMID: 18258861

Of moss and me

I recently had a look through some new papers published on moss (Physcomitrella patens) and found one published in Cell titled ‘Transcriptional control of gene expression by microRNAs’ [1]. This didn’t seem to fit the same pattern of a lot of moss papers I have read (ie in less high impact journals). This paper shows how useful moss is for studying gene expression using reverse genetics (so hopefully my PhD wont be a failure).

I think it is important to study important biological problems, such as RNA silencing and epigenetics, in a diverse range of organisms. I take this title from a short review published in Nature Medicine recently (as well as the Steinberg book) by Sir David Baulcombe (who holds a prestigious chair at Cambridge). He wrote about what we have learnt by studying things in plants (eg discovery of transposons) and their importance as a model, using his own research into RNA silencing and epigenetics as the main example [2]. I think this new moss paper goes some way to support this, but also to show the importance of this particular organism. My choice to work with Physcomitrella was because I wanted to stay in the plant world but wanted to work with something where I could knock out specific genes by homologous recombination and was simple to grow. Physcomitrella can be grown on simple agar media plates and joins the few species where genes can be knocked out.

Khraiwesh et al (2010) looked at what happened when you knocked out two Dicer-like (DCL) proteins in moss [1]. Dicer proteins in animals and plants cut long dsRNA to make small RNAs loaded onto Argonaute proteins (producing the effector complex). This effector complex is what causes the target mRNA to be cut or repress its translation. When PpDCL1a was knocked out you see a lack of microRNAs (miRNAs) being produced and the plant is restricted to its first developmental stage only. Not surprising. Strangely, when you knocked out PpDCL1b get production of miRNAs but not cleavage of the targets. It has been shown in animals, but not other plants, Dicer proteins can sometimes be used to help a small RNA in the effector complex to cut the target, as well as in miRNA or siRNA biogenesis. Very interestingly they found these targets had a lower level of expression in the mutant plants. If cleavage is not reducing the mRNA levels then what? The answer to this as many other odd questions in biology is epigenetics. Well DNA methylation at least. siRNAs have been shown to cause gene silencing but not much evidence for miRNAs doing this before. The authors went onto show the ratio of miRNA to mRNA target is critical. If you only have a little miRNA compared to mRNA target you get cleavage. If you have lots of miRNA to target mRNA it causes DNA methylation and silencing. But the methylation was only seen in the knockout so why should we care?

Finally they end the paper by showing ratio of miRNA to target mRNA is important in an endogenous gene involved with a hormone response. The plant hormone ABA mediates drought response and abiotic stress. In moss, when ABA treated you get a decrease in a transcription factor response gene. This is a target of a natural miRNA. This down-regulation is due to an increase in miRNA expression, changing the effect from cleaving the transcription factor’s mRNA to targeting its promoter for methylation and silencing expression. This study shows that miRNAs have the potential to cause DNA methylation, which had not yet been show, as well as suggesting a mechanism for it. Future studies can now go on to show the relevance of this in other systems from yeast to flies to mammals.

I end by simply saying MOSS ROCKS.

Khraiwesh, B., Arif, M., Seumel, G., Ossowski, S., Weigel, D., Reski, R., & Frank, W. (2010). Transcriptional Control of Gene Expression by MicroRNAs Cell, 140 (1), 111-122 DOI: 10.1016/j.cell.2009.12.023

Baulcombe, D. (2008). Of maize and men, or peas and people: case histories to justify plants and other model systems Nature Medicine, 14 (10), 1046-1049 DOI: 10.1038/nm1008-1046

Freedom of speech within science

Not so much research but a rant. As you may have heard the chief advisor to the government about drugs policy Prof Nutt was told to resign recently after he gave a lecture stating his views on cannabis classification in the UK and how the government (former Home Secretary Jackie Smith) did not listen to their advice on the matter and increased its classification and punishment for taking it. I do not care about the drug’s classification to be honest. I don’t take it and never have but the current Home Secretary (Alan Johnson, tipped to be future Labour leader, among a couple of others) has had a disagreement with the scientist and dismissed him after he gave a lecture stating his opinion. The reason given was he had crossed the boundary between science and politics. What? A scientist who is an unpaid advisor states his opinion based on evidence then states the government didn’t listen to them when deciding policy is crossing the line?! It sounds like he was starting a debate, using his freedom of speech to discuss topics. The government has decided no, if you don’t agree with us and you are helping us then sod off. Whether you agree with Prof Nutt or not I don’t think it matters. What matters is whether advisors are allowed to criticise the government’s policy when they are not listened to. They believe not. Alan Johnson said: ‘he was not dismissed because of the work of the council but because of his failure to recognise that... his role is to advise rather than criticise’. My PM Gordon Brown said: ‘Prof Nutt had repeatedly undermined Labour's drug message.’ I think it is ridiculous to suggest a scientist cannot talk about such things in public and suggests to me the government is being hypersensitive over these matters. Perhaps because they cannot handle anymore bad press and people going against them. After all the chances of them winning the next election are slim to none.

So far Lord Drayson (a businessman) who determines science policy in the government’s Business department and coordinates with the research councils was strongly against this when he first heard it. Lord Winston FRS, a respected scientist in the public eye and Labour peer, is against the dismissal and Prof John Beddington FRS (who recently recovered an honorary degree from York University in the Summer 2009 graduation ceremony) has spoken the most sense on the matter (http://news.bbc.co.uk/1/hi/sci/tech/8340318.stm). He is the chief advisor to the government on all things sciencey. He points out most scientific committees do not have a problem with the government. This is reassuring but and the send of an email the Home Secretary (with no background what so ever in science) has dismissed an advisor and caused two to quit in sympathy because they don’t think they can work with a government that a) doesn’t listen to them and b) forces out the head of the committee for no good reason. I think this is unacceptable from the government hence I have written to the Home Secretary and feel I should email my local MP as well. Hopefully my next post will contain more science and less ranting. It’s being planned, just need to get some pretty photos.

Ig Nobels 2009

For a few years now that I have been following the Ig Nobels as they are awarded. To continue the tradition, here is a post with the list of this year's winners:

VETERINARY MEDICINE PRIZE
Catherine Douglas and Peter Rowlinson of Newcastle University, Newcastle-Upon-Tyne, UK, for showing that cows who have names give more milk than cows that are nameless.
"Exploring Stock Managers' Perceptions of the Human-Animal Relationship on Dairy Farms and an Association with Milk Production," Catherine Bertenshaw [Douglas] and Peter Rowlinson, Anthrozoos, vol. 22, no. 1, March 2009, pp. 59-69.

PEACE PRIZE
Stephan Bolliger, Steffen Ross, Lars Oesterhelweg, Michael Thali and Beat Kneubuehl of the University of Bern, Switzerland, for determining — by experiment — whether it is better to be smashed over the head with a full bottle of beer or with an empty bottle.
"Are Full or Empty Beer Bottles Sturdier and Does Their Fracture-Threshold Suffice to Break the Human Skull?" Stephan A. Bolliger, Steffen Ross, Lars Oesterhelweg, Michael J. Thali and Beat P. Kneubuehl, Journal of Forensic and Legal Medicine, vol. 16, no. 3, April 2009, pp. 138-42.

ECONOMICS PRIZE
The directors, executives, and auditors of four Icelandic banks — Kaupthing Bank, Landsbanki, Glitnir Bank, and Central Bank of Iceland — for demonstrating that tiny banks can be rapidly transformed into huge banks, and vice versa — and for demonstrating that similar things can be done to an entire national economy.

CHEMISTRY PRIZE
Javier Morales, Miguel Apátiga, and Victor M. Castaño of Universidad Nacional Autónoma de México, for creating diamonds from liquid — specifically from tequila.
"Growth of Diamond Films from Tequila," Javier Morales, Miguel Apatiga and Victor M. Castano, 2008, arXiv:0806.1485.

MEDICINE PRIZE
Donald L. Unger, of Thousand Oaks, California, USA, for investigating a possible cause of arthritis of the fingers, by diligently cracking the knuckles of his left hand — but never cracking the knuckles of his right hand — every day for more than sixty (60) years.
"Does Knuckle Cracking Lead to Arthritis of the Fingers?", Donald L. Unger, Arthritis and Rheumatism, vol. 41, no. 5, 1998, pp. 949-50.

PHYSICS PRIZE
Katherine K. Whitcome of the University of Cincinnati, USA, Daniel E. Lieberman of Harvard University, USA, and Liza J. Shapiro of the University of Texas, USA, for analytically determining why pregnant women don't tip over.
"Fetal Load and the Evolution of Lumbar Lordosis in Bipedal Hominins," Katherine K. Whitcome, Liza J. Shapiro & Daniel E. Lieberman, Nature, vol. 450, 1075-1078 (December 13, 2007). DOI:10.1038/nature06342.

LITERATURE PRIZE
Ireland's police service (An Garda Siochana), for writing and presenting more than fifty traffic tickets to the most frequent driving offender in the country — Prawo Jazdy — whose name in Polish means "Driving License".

PUBLIC HEALTH PRIZE
Elena N. Bodnar, Raphael C. Lee, and Sandra Marijan of Chicago, Illinois, USA, for inventing a brassiere that, in an emergency, can be quickly converted into a pair of protective face masks, one for the brassiere wearer and one to be given to some needy bystander.
U.S. patent # 7255627, granted August 14, 2007 for a “Garment Device Convertible to One or More Facemasks.”

MATHEMATICS PRIZE
Gideon Gono, governor of Zimbabwe’s Reserve Bank, for giving people a simple, everyday way to cope with a wide range of numbers — from very small to very big — by having his bank print bank notes with denominations ranging from one cent ($.01) to one hundred trillion dollars ($100,000,000,000,000).
Zimbabwe's Casino Economy — Extraordinary Measures for Extraordinary Challenges, Gideon Gono, ZPH Publishers, Harare, 2008, ISBN 978-079-743-679-4.

BIOLOGY PRIZE:
Fumiaki Taguchi, Song Guofu, and Zhang Guanglei of Kitasato University Graduate School of Medical Sciences in Sagamihara, Japan, for demonstrating that kitchen refuse can be reduced more than 90% in mass by using bacteria extracted from the feces of giant pandas.
"Microbial Treatment of Kitchen Refuse With Enzyme-Producing Thermophilic Bacteria From Giant Panda Feces," Fumiaki Taguchia, Song Guofua, and Zhang Guanglei, Seibutsu-kogaku Kaishi, vol. 79, no 12, 2001, pp. 463-9. [and abstracted in Journal of Bioscience and Bioengineering, vol. 92, no. 6, 2001, p. 602.]

Looking for some science on swine flu

As a medical association in Spain has recently communicated to the press, more than anything there seems to be an epidemic of fear going on. As I am currently living in Portugal, I do not know what the situation is in other countries, but I can tell you how the situation is here. Every news bulletin starts with more news about the flu, even though no one has died here yet. It then finishes with a previously recorded warning on how one should wash hands frequently and keep 1m away from other people while talking to them, quite an ordeal in a country where the common greeting is a couple of kisses in the cheek. In addition, we have ambulances going around with the paramedics dressed as in the photo, right out of films like ‘Outbreak’. Now, most people seem to assume that because I have just graduated with a Biology degree that I should know everything there is to know about the current flu. Besides the generic question ‘So, what do you think about this flu thing?’, I have had some more specific questions, from ‘I heard that it is impossible for this combination of genes to happen in nature, surely this must have been created by evil scientists with corporative interests?’ to ‘This virus is called H1N1, the 1918 pandemic virus was also H1N1, are we all going to die of Spanish flu?’. I try to explain people that at uni we are taught how to get informed about things, rather than becoming specialist on every single biological problems; and that frankly I am so fed up of hearing about this flu that I couldn’t be bothered to look it up. However, I think the hysteria might be affecting me, probably because I’ve just read the book Blindness (if you have read it/watched the film you will know why). So I decided to interrupt my holidays-away-from-science to read a bit about this topic, and thought I would tell you some of my findings.

My first port of call was the website of the World Health Organisation, usually a reliable source for information. I was pleasantly surprised by the lack of over-dramatic information. There is of course a whole section dedicated to this flu, right in the home page, but the answers given in the FAQs seem quite sensible. It tells us that 2009 A(H1N1) is spread as the normal flu, and that the current worries as based on the fact that being a virus which never circulated in humans before, there is no, or very little, immunity. In addition, as stated in their website, the virus is spreading fast in young people (10-45), from a majority of mild cases to some serious illnesses, the majority of which in patients with underlying conditions. The recommendations of the WHO are to take pain killers and drink loads of fluids if you have the flu. And only contact the medical services if you have serious symptoms like shortness of breath, or if the fever lasts for longer than 3 days. Quite unlike what recently happened in Portugal. When a man who had travelled to Britain was diagnosed with this flu, he saw his house invaded by doctors who quickly took him away to a hospital and confined the whole family to their house. Without telling them what to do or when they could leave.

Anyway, I then thought it would be a good idea to read some information from journals, a bit more scientific and hopefully less dramatic source of news. However, as you know, I am currently unable to access most papers, as I am in between universities and am yet to be provided with a username in my new institution. So what follows is based on a couple of papers I could access, and is by no means comprehensive.

As an introduction, some information from the Virology handouts we were given last October. Influenza viruses are RNA viruses that have a segmented genome (8 segments/genome). The glycoprotein spikes, haemagglutinin (HA) and neuraminidase (NA) are important in the entrance of the virus in the host cells. There are 15 known HA and 9 NA serotypes, and their combination provides the name that we see for the viruses such as H1N1. Birds and pigs are important reservoirs for genetic and antigenically diversity and reassortment in these viruses. In particular pigs, as their cells can be infected by both avian and human influenza viruses, making them nice ‘mixing pots’.

In July this year Garten et al., published a paper in Science in which they characterised the 2009 A(H1N1) virus both antigenically and genetically. They start the paper by giving a small introduction on the influenza pandemics of the last century, and how we got from those to the current 2009 A(H1N1). Then it goes on to characterise the actual viruses. The closest ancestral gene for each of the eight segments seems to have a swine origin, although having originally come from avians and sometimes humans in different occasions. This is quite interesting, as swine influenza viruses had not, until now, caused much disease or been incredibly good at human-human transmission. As the authors point out, this virus probably had been circulating for a while in pigs, unnoticed due to lack of monitoring.

Analysis of the virus genome showed that none of the molecular signatures of increased transmissibility or virulence of A(H1N1) viruses can be found in this strain, and that functionally important receptor binding sites on HA do not differ from classic swine influenza viruses. No markers were also found that indicate adaptation to the human host or features of previous pandemic virus. The main important difference seems to be a genetic marker for resistance to adamantine antivirals, but the virus still seems to be sensitive to the other type of antivirals, the neuraminidase inhibitors. So, this study concludes, we must be worried about this virus firstly because we don’t really know what makes it good at replicating in humans, and secondly because it has a genetic composition not seen before, so we don’t really know what to expect.

I then went on to read a couple of papers regarding studies done in mammal models such as ferrets (apparently classic models for influenza studies) and mice.

The first study was published in Science also in July, by Maines et al. This post is becoming quite long already, so I’ll just summarise their results (based on 3 independently isolated 2009 A(H1N1) viruses as compared with a seasonal influenza strain). They basically inoculated 106 p.f.u in ferrets and monitored different indicators such as body weight, viral titres, direct and indirect transmissibility, etc. The main conclusion from the study was that 2009 A(H1N1) caused higher morbidity (weight loss, etc, depending on which virus isolate), showed, unlike seasonal flu, high viral titres in the lower respiratory tract or the intestine, but that it was less efficient at indirect transmission (putting healthy ferrets in cages near to inoculated animals).


The second animal study I looked at was published online by Nature, in what they call a ‘near final version’. They also used virus isolated from infected humans, though not all of which were the same as in the Science paper. They studied the effect of these viruses in mice, macaques, ferrets and pigs, though not all of the virus isolates seem to have been used in all of the experiments. They also looked at more indicators, such as pro-inflammatory cytokines, lung pathology, etc. In ferrets the results were similar to those of the Science paper, except that this time indirect transmission was successful. In mice and macaques, the 2009 A(H1N1) virus seems to be worse than the seasonal virus (at least one of the isolates that they seem to mostly use throughout, CA04) although according with which criteria varies with the model. The inoculated pigs were asymptomatic, explaining why the virus had not been noticed before in this animal.

So, what are the conclusions I could get, overall, from these three studies? Overall it seems that the 2009 A(H1N1) virus is worse than the seasonal influenza viruses in mammalian models. However, how much worse is probably hard to say. I am not familiar at all with viral studies, so it is hard for me to critically analyse these studies, but it seems to me that in most cases the n numbers were quite small (n=3 in some cases in the Nature study), but this is perhaps not surprising, as they must have been quite quick studies, considering the current need to understand this new virus. And, as it is probably the case in most animal studies in general, I reckon the doses used were probably much higher than what humans would be exposed to in the real world. In addition, there is always the question on how well do these animal models really reflect what happens in humans.

In any case, it seems that so far these studies indicate that 2009 A(H1N1) is, in these models, worse than seasonal flu. Whether it is as bad as some of the pandemics that have been affecting us in the last 100 years it is unknown. The other worry is of course that the virus might evolve into something much more dangerous. Though all the virus isolates obtained so far tend to be quite similar, 5 minor genome variants have already been detected. Restricting the spread of the virus, even if it is not so bad right now, might be a good way of preventing it from acquiring any more deadly features. Whether this data justifies the measures being taken by most government, that is really, as PHD comics puts it so well, beyong the scope of my area of study. I will leave that to those who study the spread of pandemics and know of population health. I think we have had enough speculation for one disease.

Garten, R., Davis, C., Russell, C., Shu, B., Lindstrom, S., Balish, A., Sessions, W., Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J., Smith, C., Emery, S., Hillman, M., Rivailler, P., Smagala, J., de Graaf, M., Burke, D., Fouchier, R., Pappas, C., Alpuche-Aranda, C., Lopez-Gatell, H., Olivera, H., Lopez, I., Myers, C., Faix, D., Blair, P., Yu, C., Keene, K., Dotson, P., Boxrud, D., Sambol, A., Abid, S., St. George, K., Bannerman, T., Moore, A., Stringer, D., Blevins, P., Demmler-Harrison, G., Ginsberg, M., Kriner, P., Waterman, S., Smole, S., Guevara, H., Belongia, E., Clark, P., Beatrice, S., Donis, R., Katz, J., Finelli, L., Bridges, C., Shaw, M., Jernigan, D., Uyeki, T., Smith, D., Klimov, A., & Cox, N. (2009). Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans Science, 325 (5937), 197-201 DOI: 10.1126/science.1176225

Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, Gustin KM, Pearce MB, Viswanathan K, Shriver ZH, Raman R, Cox NJ, Sasisekharan R, Katz JM, & Tumpey TM (2009). Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science (New York, N.Y.), 325 (5939), 484-7 PMID: 19574347

Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe, S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M., Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H., Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Takahashi, K., Warshauer, D., Shult, P., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K., Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M., Sugaya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K., & Kawaoka, Y. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature DOI: 10.1038/nature08260