The gifts under the tree
“O Tannenbaum, o Tannenbaum, Dein Kleid will mich was lehren: Die Hoffnung und Beständigkeit Gibt Mut und Kraft zu jeder Zeit! O Tannenbaum, o Tannenbaum, Dein Kleid will mich was lehren!” From O Tannenbaum, a German folk song
“O Christmas tree, O Christmas tree, your appearance will teach me something: Your hope and durability is courage and strength at any time! O Christmas tree, O Christmas tree, your appearance teach me something!” Translation of O Tannenbaum, a German folk song
Nature is remarkable in its resilience.
Here, temperatures have plummeted, heavy snow has fallen, merciless winds have whipped. Only the hardiest of beings persist under such inclement conditions.
While their boughs droop under the weight of the snow, the conifers stand steadfast against the conditions. With their evergreen branches draped with a mantle of white, they stand like stoic shepherds minding their wintry flocks.
The conifers seem to fare well, even in such an adverse environment. In the short days of winter, conifers convey a strong message of survival against the odds. No small wonder that we welcome them into our homes during the holiday season – an emblem of our shared capacity to wait out the unforgiving challenges of winter – to see it through to another year.
The conifers, which include the spruces, pines and firs, comprise a division within the larger gymnosperm group of seed plants. They are dominant tree species in many ecosystems, particularly those of the boreal forest, or taiga. Over their 350 million year evolutionary history, conifers have emerged with a suite of traits that enable them to contend with unsympathetic winter weather.
In preparation for the winter, a hard bud is established at the tip of each branch at the end of the growing season. Within the bud, the cells that will give rise to new growth next year, the cells of the shoot apical meristem, are shielded against the conditions by a hardened outer casing. There the cells of the shoot apical meristem remain relatively inactive over the winter months – in a state of hibernation-like dormancy – until prevailing conditions are suitable for them to be active again.
The needle-like leaves of the conifers are well-suited to winter existence. With their limited surface area, and their toughened outer cuticle, they are relatively resistant to water loss. Their cells fill with solutes, particularly sugars, to prevent freezing. Importantly, their cells also fill with sunlight-blocking pigments to limit the amount of photosynthesis that takes place – as little, if any, is needed during the winter months. Similarly, the chemistry within the water-conducting cells of the stem changes to curtail water transport, and to prevent freezing-induced embolism that would prevent water transport again in the spring.
All told, evolution has equipped conifers with the capacity to ride out the adversity of winter. Mutation and natural selection have provided conifers with gifts that literally sit under the tree, support it, as it persists against this adversity.
Simultaneously, evolution has provided conifers with another gift.
The genome is the DNA-based set of instructions for building an organism. The instructions are genes, written in the four letter code of the nucleotide bases – A, C, G and T. The nucleotide bases are arranges as pairs along the DNA double helix. The overall size of a genome is measured in base pairs.
In each cell of our bodies, you and I have a genome that contain 6 billion base pairs of DNA organised on 23 pairs of chromosomes. Sometimes you will see this number halved, to 3 billion base pairs, in considering just the complete set of 23 chromosomes, instead of the 23 pairs that reside in each cell. For the purposes of our discussion here, we will consider this number, 3 billion, as it is most commonly used in scientific papers.
In contrast to the human genome, with its 3 billion base pairs, conifer genomes are huge. Conifer genomes vary in size from approximately 20 billion to 40 billion base pairs. That is, they are around 10 times larger than the human genome.
How can this be?
On the face of it, it may seem strange that an organism that we consider to be as complex as a human being should have a genome so much smaller than that of a tree. But this would be a rather simple view. While we go about our lives in a different manner, it is merely different – not necessarily more or less complex. As they are literally rooted in one place, trees have to contend with a variety of environmental insults on a daily basis, throughout their long lifetimes, that we do not have to contend with.
There is no reason to think that the number of genes contained within the genome would need to be any more numerous for us to go about our business thank for a tree to go about its business. In keeping with this, the number of genes contained within our genome is about 21000; whereas, the number of genes estimated within the best characterised conifer genome, Norway spruce, is not that different at around 28500. This is not far different from one of the smallest characterised plant genomes, that of mouse-eared cress, Arabidopsis thaliana, which has around 27500 genes contained on 135 million base pairs of DNA.
So what accounts for the large size of the conifer genome?
Genomes can grow by a number of mechanisms. The number of genes is just one way. There evidence suggests that this is not the way that the conifer genome got so large. Another way that a genome can increase in size is by multiplying the number of chromosomes within a cell.
Chromosomes are the way in which DNA is organised in cells. We have 23 pairs of chromosomes in each cell. The mouse-eared cress has 5 pairs of chromosomes in each cell. Conifers have 12 pairs in each cell.
Some plants have increased genome size by having more pairs of chromosomes. These additional pairs of chromosomes arose from what is known as a whole genome duplication event. Whole genome duplications occur periodically in evolution. For instance, whole genome duplication in the relatively recent history of cereal crops, like wheat, has played an important role in the evolution of those crops.
There is evidence for whole genome duplication in the Norway spruce genome. This said, this duplication is ancient. It appears to predate the evolutionary split of gymnosperms (like conifers) from the other major group of land plants, angiosperms. Angiosperms include all the broad-leafed plant species, from apples to zinnias. Angiosperms and gymnosperms followed different evolutionary trajectories some 350 million years ago. The fact that the whole genome duplication event in Norway spruces predates this date indicates that whole genome duplication does not account for the large conifer genome size.
Given that neither genome duplication nor gene numbers account for the remarkable size of the conifer genome, there must be another, more devious mechanism. And there is.
Conifer genomes seem to have grown on account of the activity of DNA that might best be described as “selfish”. This DNA appears, on the face of it at least, to exist for no other purpose than to produce more copies of itself.
The selfish DNA in question is known as transposable elements.
Transposable elements are remarkable pieces of DNA. The type of transposable elements that enlarged conifer genomes function by replicating themselves, and then inserting the replicated copy back into the genome. The replication process involves converting the transposable element DNA into a copy made of a different nucleic acid, RNA. This occurs as a normal part of the function of the genome, as gene instructions are read out, or transcribed, as RNA.
Under most circumstances, RNA is used as instructions to make a protein and then degraded. In the case of the transposable element-derived RNA, some parts of the RNA are made into proteins. On of these proteins is very cunning – it converts the RNA back into DNA. As it is involved in converting the transcribed RNA back into DNA, this protein is called reverse transcriptase. The other protein is equally cunning, in that it recognises the DNA made by the reverse transcriptase, and integrates it back into the genome. For this reason, the protein is called an integrase. The integrase recognises long terminal repeats of nucleotide bases found at the outer edges –the borders if you will – of the DNA that has been made from the RNA. It grabs hold of these sequences and uses them to integrate the DNA copy back into the genome. When it does so, a new piece of DNA has been added to the genome.
Because the replication of the transposable element involves the retrograde conversion of RNA back to DNA, these kind of elements are called retrotransposons.
Retrotransposons are found across diverse taxa, from humans to flies to trees. There are two major types of retrotransposons found in plants like conifers. They are referred to as Ty1 or copia, and Ty3 or gypsy.
There are at least two major consequences of retrotransposon activity. One is obvious – as they replicate, and are inserted back into the genome, they will increase genome size. The other outcome is maybe a little less obvious. As they insert into the genome, the retrotransposons are somewhat indiscriminate about where they are parked. Consequently, retrotransposons may be inserted into functional genes, and therefore have the capacity to disrupt the function of that gene. If this makes the gene dysfunctional, that can be problematic.
On account of the disruptive function of retrotransposons, plants have evolved mechanisms to counteract their effects. One mechanism involves shutting the genes of the retrotransposon off. That is, the plant disables the retrotransposon by shutting down the reverse transcriptase and integrase genes. It does this by chemically silencing the retrotransposon DNA, the genes, that encode those two proteins. The other mechanism involves purging the newly inserted retrotransposon DNA during the production of pollen and eggs. The process of egg and pollen production involves the cellular process of meiosis. During meiosis, recombination of chromosomes involves a form of “genome surveillance”, which can function to purge the genome of bits of DNA that don’t match up between pairs of chromosomes. Between these two mechanisms, plant contain the activity of retrotransposons and their after-effects.
Remarkably, conifers seem to be impaired in their ability to completely silence retrotransposons, and to purge them once they have replicated and inserted. Evidence suggests that conifers may be lacking certain genes that encode proteins that function to monitor and curtail retrotransposon activity. Regardless, the net result is that retrotransposons have been building up, slowly, in conifer genomes.
In fact, when retrotransposons act in most other plants, their evidence is visible over even short time scales – but are then purged. By contrast, in conifers, retrotransposons work slowly. For example, evidence of significant retrotransposon activity can be seen in periods of less than 5 million years in different rice species; whereas, retrotransposon differences are only observed infrequently in different spruce species over a period of 13-20 million years. This likely explains why conifer genomes are so similar, despite their large size.
One of the major impacts of the retrotransposon activity in conifer genomes is that they have inserted into genes, rendering them dysfunctional. Consequently, conifer genomes tend to carry a lot of “trashed” genes – ones that no longer can do the job they were originally intended to do. These are called pseudogenes.
Pseudogenes abound in conifer genomes. Fortunately, they are compensated for by equivalent functional genes that continue to carry out the function that was destroyed by the retrotransposon. Nevertheless, this has a consequence for conifer genome size. The genome continues to carry these dysfunctional copies. If they confer no selective disadvantage on the plant that carries them, there is no way to purge pseudogenes – they just stay along for the ride. This is now DNA that could be viewed as a form of selfish baggage – conferring no obvious advantage but getting replicated and transmitted from one generation to the next.
Thus, between retrotransposons themselves, and the pseudogenes they create, conifer genomes have taken the route to “genome obesity”. It would appear that is has been a slow route. DNA accumulated over tens or even hundreds of millions of years. This occurred in the early conifers, so that the conifers the we know today share many of the marks of this expansion in the same locations, with the order of genes retained on their shared 12 pairs of chromosomes.
Here’s a hypothesis, a gift, if you will. Perhaps there is a two-fold advantage to retrotransposon activity in conifers. The first is that it generates genetic diversity. For long-lived species that produce the next generation infrequently, there might be advantages to having a large standing pool of genetic variation. If the neighbours you pollinate are related to you, better they should be carrying some genetic diversity that you don’t possess. Even though retrotransposon activity can destroy gene function, it can also create genetic novelty – novelty that may confer an advantage to the next generation.
Aside from this, it may be that having a large genome confers some advantage that we cannot fathom at this point in time. Conifers include some of the longest lived, largest, and tallest organisms on the planet. Might it be that having a large genome contributes to some of those grand features? With the new tools of genome biology, we might begin to test this and related hypotheses.
The take home message is that it is still too early to tell whether the DNA we deem “selfish” or “junk” or otherwise useless is, in fact, any of these things. It may truly be the gift that keeps on giving.
At this time of the year, as you marvel at the capacity of conifers to contend with the prevailing conditions, and as you bring them into your homes to add to the festive spirit, take a moment to ponder not merely the gifts that sit beneath the tree, but the gifts found within it. Evolution has bestowed on conifers an incredible capacity to survive - so evident from the outside. It has also bestowed a stunning molecular "gift" - something hidden within. Sometimes those gifts that create the greatest sense of wonder are those that require a little more work, a little more searching within.
Images: All photographs by Malcolm M. Campbell.
Bennetzen JL & Kellogg EA (1997) Do plants have a one-way ticket to genomic obesity? The Plant Cell 9: 1509-1514
Devos KM, Brown JK, & Bennetzen, JL (2002) Genome size reduction through illegitimate recombination counteracts genome expansion in Arabidopsis. Genome Research 12: 1075-1079
Friesen N, Brandes A, & Heslop-Harrison J S P (2001) Diversity, origin, and distribution of retrotransposons (gypsy and copia) in conifers. Molecular Biology and Evolution 18: 1176-1188
Morse AM, et al. (2009) Evolution of genome size and complexity in Pinus. PLoS ONE 4: e4332
Nystedt B, et al. (2013) The Norway spruce genome sequence and conifer genome evolution. Nature 497: 579-584
Pavy N, Pelgas B, Laroche J, Rigault P, Isabel N, & Bousquet J (2012) A spruce gene map infers ancient plant genome reshuffling and subsequent slow evolution in the gymnosperm lineage leading to extant conifers. BMC Biology 10: 84