Hot Arabidopsis and its triplet trouble

It was the green worms that gave it away: while studying a wild strain of Arabidopsis thaliana and subjecting it to warmer than normal conditions, Dr Sureshkumar Balasubramanian and colleagues noticed that the plants produced weirdly misshapen leaves and never progressed to the flowering stage.

They named the phenotype “greenworms”, which the leaves resembled, and set about trying to find out the genetic basis for the defect.

What they found was a dramatic expansion of a TTC/GAA triplet repeat when compared to other strains of Arabidopsis, including the sequenced strain Columbia (Col-0).

The wild strain, called Bur-0 and native to the cool climes of Ireland, had more than 400 repeats in an intron of a gene which codes for an enzyme called isopropylmalate isomerase, compared to 23 copies in Col-0.

The gene, now called isopropylmalate isomerase large subunit 1 (IIL1), is involved in amino acid biosynthesis. The triplet repeat expansion causes a reduction in IIL1 expression at high temperatures, resulting in wormy leaves and a general failure to thrive.

At normal temperatures the phenotype is not apparent, which is probably why the defect has survived. “Obviously, having this repeat is detrimental to the plant, so if it experiences hot temperatures, evolutionarily it should have been removed,” Balasubramanian says.

“The fact that it remains tells you that it is unlikely to have experienced those hot temperatures. [Artificially] providing those different conditions means you can reveal hidden genetic variation and figure out additional variation that leads to phenotypic differences that might be helpful in some conditions and not so helpful in others.”

Triplet repeat expansions can be very unhelpful indeed. In humans, they are the cause of a range of disorders such as Huntington’s disease, Friedreich’s ataxia and Fragile X syndrome.

These conditions are very difficult to study at a molecular level, which is why Balasubramanian and his colleagues believe the Arabidopsis finding may prove it to be a model organism to study not just triplet repeat disorders but genetic anticipation – in which severity increases with each generation, as is seen in Huntington’s – as well.

In humans, triplet repeat expansion disorders come in two main forms: Disorders where expansions result in poly amino acid stretches (typically polyglutamine or in some cases polyalanine) like Huntington’s and a variety of spinocerebellar ataxias, and disorders where there is no change in amino acids, like Fragile X, Fragile XE mental retardation and Friedreich’s ataxia.

In the former, the expansions occur in the exons of CAG or GCN codons, leading to changes in levels of the amino acid glutamine or alanine. Typically, there need to be 60 or more trinucleotide repeats to give rise to disease.

In the latter, the repeats occur in the non-coding regions, and in these cases the expansion is massive. It is thought that these expansions interfere with the work of DNA polymerase, leading to a reduction in transcripts, which leads to a reduction in protein and hence giving rise to disease.

The triplet repeat expansion Balasubramanian and colleagues have found in Bur-0 is one of these latter varieties, which is helpful. “Because these expansions are so massive, it’s difficult to engineer them genetically to study,” he says.

“With the polyglutamine disorders like Huntington’s, people have been able to replicate the genomic instability, for example at Nancy Bonini’s lab [at the University of Pennsylvania], where they published a paper in Science a couple of years ago showing that you can mimic a similar situation by transgenesis, as far as the coding region disorders are concerned.

“But the massive ones are almost impossible to engineer – it basically gets very difficult and unstable. So in the absence of other systems, we feel that this one provides a very nice tool for study. The plants can be grown in three months.”

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Natural variation

Balasubramanian’s co-authors on the Arabidopsis paper, published recently in Science, include his wife, Sridevi Sureshkumar, graduate students Marco Todesco and Korbinian Schneeberger and a masters student, Ramya Harilal, who worked with him in Professor Detlef Weigel’s laboratory at the Max Planck Institute for Developmental Biology in Germany, where Balasubramanian did post-doctoral work after finishing his PhD.

He obtained his PhD from the University of Zurich in 2002, where he worked out the function of an Arabidopsis gene called NOZZLE – mutations cause the gene to make an ovule that looks like a nozzle – in the laboratory of Professor Kay Schneitz, before pursuing his further interest in natural variation at Max Planck.

Balasubramanian is now happily ensconced at the School of Biological Sciences at the University of Queensland, where he works alongside others in the Ecological and Evolutionary Functional Genomics initiative. This group is free to research unrestricted by system: it is the fundamental evolutionary processes that are important, rather than the system itself, he says.

“Here, we have five different groups working on completely different things, including Drosophila, sunflowers, marine organisms, insects and plants and so on. To gain evolutionary insights, very often people study the same pathway in two different species, so in an evolutionary sense, you are looking at two evolved pathways.

“Whereas if you are studying variation at the micro level, within species, as we do, we are looking at evolutionary processes, things that are actually happening. Among these some may lead to evolutionary changes and some may not.

“It’s a very diverse group, and while I have specific expertise in developmental genetics and biology in Arabidopsis, I am also benefiting from the ecological and evolutionary knowledge arising out of other systems studied here.”

Weigel’s laboratory in Tubingen concentrates on understanding the molecular reasons for how and why different phenotypes arise, research that Balasubramanian continues at UQ.

“My interest is in studying the decision of when to flower, how and when that decision is made,” he says. “It’s obviously important in the lifecycle of a plant and it is controlled by a variety of factors, including the endogenous developmental stages of the plant as well as the environmental conditions, especially things like day length and temperature and so on.

“We understand a lot about how day length regulates flowering time, but we don’t really understand how small changes in temperature actually modulate flowering time. We do know to some degree that it does affect it but we don’t know the molecular mechanics of it. That’s what my lab wants to find out.”

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Irregularly impaired leaves

This is partly what led Balasubramanian and his colleagues to look at the greenworm phenotype, now renamed “irregularly impaired leaves” (iil) to suit the IIL1 gene. (“Geneticists like to name their mutants, although I don’t know how much sense it makes to others,” he says. “But for the students, and for us, it’s fun.”)

“The initial idea was that we looked at strains of Arabidopsis from different parts of the world. These strains have obviously adapted and have been growing in those conditions for a long time, and presumably some of them would have acquired the genetic changes that are required to perform better under those conditions. The idea is that if you provide them identical conditions, there will be differences, and the differences that you see will be mostly because of variations at the genetic level.”

Arabidopsis is what is called a facultative long-day flower: in the presence of long days, that is 16 hours of light, it flowers much earlier than during short days, he says. “When long days set in spring or in early summer, that gives the plants a clue and they flower early.”

However, even without a long-day clue, by artificially creating short days of about eight hours of light and then increasing the temperature from 23 to 27 degrees, researchers can induce flowering to the same extent as long-day does.

“In real life you obviously have the day length as well as the temperature and everything comes together and there is a cumulative decision made,” he says. “For research, we need ways of specifically looking at individual responses in which we can identify what are the molecular mechanics that underlie it.

“We still continue to work on flowering time, and I recently got an ARC grant to work on looking at temperature responses and flowering. We are studying Arabidopsis but we are obviously trying to extend our study to crop species, in our case to sorghum.”

Balasubramanian is actively looking for students to assist him and the rest of the ‘SKB lab’ on this quest.

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Population dynamics

In the meantime, work continues on the wild strain with the triplet repeat. This strain is native to Ireland and Balasubramanian intends to explore the population dynamics of repeat lengths in this strain with one of his collaborators, Dr Charles Spillane from Ireland’s University College, Cork, whom he met in Switzerland during his PhD days.

Spillane is now looking at C. elegans to see if similar kinds of defect occur, with the notion of eventually pooling resources for further exploration.

“We don’t really know at an evolutionary, population and genetic level what are the forces that operate and how it is maintained in populations,” Balasubramanian says.

“This provides an opportunity to study that as well. We plan to go back to the local populations and study the population level dynamics, across generations.”

In the Science paper, the researchers demonstrated that the triplet repeat was responsible for the iil phenotype by conducting mutagenesis screens. And by using the mutagenic chemical ethyl methane sulfonate (EMS), they have identified mutants in which the phenotype is suppressed, even when there is a massively expanded repeat.

In reality, he says, there must be second-site suppressors – something else that modifies the effect. “Now the idea is that if you identify any modifier that can help the DNA polymerase to go through these difficult stages, that would be of immense value.

“This is one of the advantages the system provides, and we just have to see how we can tailor it to gain more. Obviously there is an interest in a medical application but the basic knowledge in terms of understanding at the population level is also important.

“There is a myth that these diseases are only found in humans. Common sense says you should see it in other species too – it’s just that other species are not being looked at the molecular level. I think that’s where working with systems like Arabidopsis is extremely helpful as you have mutants in almost every gene.

“We can transform the genes from other species and see if it can rescue the phenotype. Effectively, we can use Arabidopsis like a toolkit.”