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Mystery at the ends of chromosomes

Researchers from Ceitec MU have determined what telomere sequences in plants of the genus Allium look like.

Left to right: Jiří Fajkus, research team leader; Vratislav Peška, unusual telomere project leader; Petr Fajkus, lead author of the study.

Researchers at the Mendel Centre for Plant Genomics and Proteomics, Ceitec MU recently revealed one of the secrets of garlic, onion and other plants in the Allium genus. In comparison to other plants, they have unusual telomere sequences. Telomeres are sequences at the ends of chromosomes, which ensure the stability of the whole genome.

Chromosomes are complex structures consisting of proteins and linear DNA, which carries the organism’s genetic information. A healthy cell usually contains a given number of chromosomes of a certain shape and length. It is quite usual for chromosomes to get damaged or completely broken, but cells have mechanisms that can repair the sustained damage. This is where telomeres come into play: they help the cell distinguish between the natural ends of chromosomes and unrepaired broken ends using a specific sequence of telomere DNA and linked telomere proteins.

Telomeres are thus remotely similar to aglets that show you which ends need to be tied together if your shoelaces get torn and make sure that the actual end does not fray and works the way it is supposed to work. Just like the rest of genomic DNA, telomere sequences contain the bases thymine (T), adenine (A), guanine (G), and cytosine (C), but in specific short and repeating patterns. “As far as we know, it is always a six-nucleotide pattern TTAGGG in humans and other vertebrates and seven-nucleotide repeating sequence TTTAGGG in most plants,” says Jiří Fajkus, the leader of the research team. “As opposed to vertebrates, however, there are already three known evolutionary lines of plants with different patterns.”

German experts first discovered plants that lacked the typical telomere sequence in 1995; the plants in question were from the Alliaceae family. But until now, nobody has described what their telomeres actually look like. The first alternative plant telomere pattern was identified in 2000–2003. Incidentally, it was TTAGGG, the same telomere pattern that is present in humans and other vertebrates.

As Vratislav Peška, the leader of the research project for unusual telomeres, explains, “This pattern can be found in the evolutionary line that includes garlic and onion, but also in other genera, for example in plants from the Iridaceae and Asparagaceae families; it is also found in Aloe vera, to give an example from among well-known plants. The plant families that underwent these changes were clearly described by my former supervisor, Eva Sýkorová, around 2003. However, the telomeres in the Allium genus as such – that is, garlic, onion, leek, chives, etc. – have only been described this year. As opposed to the most common pattern, the sequence unit of these plants contains twelve nucleotides: CTCGGTTATGGG. Last year, we described one more alternative telomere in Cestrum elegans, a South American ornamental shrub, whose pattern contains ten nucleotides – TTTTTTAGGG.” Peška adds that one more unusual telomere with a TTCAGG/TTTCAGG pattern was described in 2015 in cooperation with Jiří Fajkus’s team in Genlisea, a North American carnivorous plant genus. Thus far, those are the only known telomere exceptions in the Embryophyta (“land plant”) subkingdom, but it is expected that other exceptions could be found in algae.

The knowledge of the end part of chromosomes can be useful, for example, when genetically modifying plants. “Plant breeders try to endow the garlic genome with resistance to rust by cross-breeding related plants of various species and genera,” says Petr Fajkus, the lead author of the study. “This process could be made significantly simpler and quicker by creating a chromosome that would already contain this resistance. As long as the end telomere sequence remained unknown, these techniques could not be applied to the genus Allium.

Rather than having an immediate practical impact, however, the extraordinary findings of the research team, whose studies on alternative telomeres were published in 2015 and 2016 in The Plant Journal, a prestigious scholarly journal, are important for further research of telomere evolution and function and their role in the emergence of new species. “From an evolutionary point of view, garlic is the youngest in the order where different telomeres are present. This means that in the deep past, there must have been at least two telomere sequence changes in the garlic line. However, each change has a huge impact on the organism: the whole way in which cells function must adjust to the change, especially the proteins that can recognise the telomere sequence and thus ensure their protective function. What we would like to find out is how the plants in the Alliaceae family survived this change and why some evolutionary lines seem to be more susceptible to these changes (and perhaps also more resistant to them), while elsewhere they would just become an evolutionary dead end,” says Professor Fajkus.

Based on the latest findings, his team will now focus on telomerase, an enzyme that basically creates telomeres. Telomerase consists of a protein that ensures reverse transcription of genetic information from RNA to DNA and an RNA subunit, which serves as a template for the telomere sequence. “We think that the changes happened at the level of the RNA subunit,” explains Petr Fajkus, adding that in plant biology, this subunit has only been described in a single plant species, thale cress.

“We are, of course, unable to tell precisely what happened in the evolution of garlic and when, but we can deduce it by comparing, say, the onion RNA subunit with other plants. That requires further research, however. We want to find out what the molecular cause of the change was and how the cell adapted to that change, but we are only at the beginning,” adds Vratislav Peška

Besides shedding new light on the telomere mystery in garlic and Cestrum elegans, the Ceitec MU researchers also helped establish a new telomere research method. This is because the plants they examined have a genome that is many times larger than the human genome, even though it has a smaller number of chromosomes (and therefore telomeres). Consequently, to read the whole genome when looking for telomeres would require a lot of time and money with an uncertain result.

The genome of the Cestrum elegans shrub, with its 9–12 billion bases in one set of eight chromosomes, is approximately three or four times bigger than the human genome (approx. 3 billion, chromosomes). The genome of the common onion, which belongs to the Alliaceae family, is even bigger (16 billion, 8 chromosomes), and the genome of wild garlic is approximately ten times bigger than the human genome (approx. 30 billion nucleotides, 7 chromosomes). The telomeres, which are the focus of the research, thus represent only a small part of these large genomes.

As Vratislav Peška describes: “This is why we combined two approaches: massive parallel sequencing (next generation sequencing - NGS) and end-specific DNA degradation. Rather than complete genome sequencing – reading the whole genome – we only looked for repeats, that is, sequences that occur many times within the genome, and we hoped that the telomere sequence would be among them. However, there are usually a number of repeats in a genome; in fact, they are responsible for the difference in genome sizes in different species. We therefore took a sample of the same DNA that was also being sequenced and we used an enzyme that can gradually eliminate the ends of chromosomes. By comparing the results, we found out which sequences were eliminated by the enzyme in the second sample and therefore could be telomeres. Afterwards, we verified the result using traditional approaches from telomere biology, such as terminal restriction fragments analysis, fluorescence in situ hybridization (in situ meaning directly on the chromosomes), and most importantly sequence analysis of the products that are created by the telomerases of the plants directly in the test tube.Peška adds that they used this approach to examine eleven plant species and finally arrived at a sequence that was the same for all eleven species of the genus Allium that were analysed.